BR112012004268B1 - processes for producing silk dope - Google Patents

Abstract

PROCESSES FOR PRODUCTION OF SILK DOPE The present invention relates to methods of producing silk dope which comprises silk proteins with a coiled-coil structure such as, for example, bee silk proteins. Silk proteins are obtained from cells that produce them, solubilization of proteins by their contact with a surfactant or an ionic liquid and concentration of proteins to produce silk dope. Proteins can be used for various purposes, for example, in the production of personal care products, plastic, textiles and biomedical products.

Description

FIELD OF THE INVENTION

The present invention relates to methods of producing silk dope comprising silk proteins with a coiled-coil structure such as, for example, bee silk proteins. The silk dope can be used for various purposes, for example, in the production of personal care products, plastics, textiles and biomedical products.

BACKGROUND OF THE INVENTION

Silks are protein fibers produced by a wide variety of insect and spider species. The silk of the domesticated silkworm, Bombyx mori, has been used as a suture biomaterial for centuries. Various efforts to clone and express silkworm or spider silks in transgenic systems have proved to be a Herculean task. The large sizes and highly repetitive sequences of these silk genes make them difficult to express outside the specialized silk glands, and lead to low 20 protein yields.

Although silkworm cocoons and spider webs are the best known silks, other species can produce silks that are more suitable for transgenic production. Bee larvae (Apis mellifera) weave silk cocoons in which 25 they soak. Bee silk is encoded by four small fiber genes (approximately 30 kDa each) and non-repetitive (Sutherland et al, 2006). Homologous sets of four genes have also been found in long-hornet hornets, bulldog ants, weaver ants, wasps and 30 Asian bees (Sutherland et al., 2007; Sezutsu et al., 2007; Shi et al., 2008; WO 2007 / 038837).

A classic X-ray fiber diffraction study demonstrated that bee silk contains helical proteins assembled in a coiled-coil conformation, most likely a coiled-coil tetrameric structure (Atkins, 1967), with the four strands probably corresponding to to four different silk proteins. Bioinformatics techniques predict that each of the sequences of bee silk protein contains 60-68% coiled-coil (Sutherland et al., 2006). Silk threads can be removed by hand from the silk glands of bee larvae. These threads are less strong, but more extensible and rigid, than the silk fibers of the silkworm (Hepburn et al., 1979).

Shi et al (2008) recently reported the recombinant production of Asian bee silk (Apis cerana). The four silk proteins of A. cerana were expressed in a soluble form in Escherichia coli with yields of 10-60 mg per liter of yeast. Several experimental techniques were used to characterize the structure and interactions of proteins and low concentration (0.03 to 0.2% by weight). These conclusively demonstrated that neither the individual proteins nor a mixture of four proteins had close tertiary compaction in solution. The proteins existed as loosely associated monomers or dimers and had a predominantly random coil conformation, with little α-helical structure.

There is a need for additional methods to produce silk dope by recombinantly expressed silk coiled-coil proteins that can be used to manufacture a wide variety of products.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that surfactants and ionic liquids can be used in a process to produce silk dope comprising coiled-coil silk proteins.

In a first aspect, the present invention provides a method for the production of silk dope, the method comprising: i) lysis of cells that produce one or more silk proteins, ii) solubilization of silk proteins by their contact with a surfactant or an ionic liquid, and iii) concentration of silk proteins to produce silk dope, in which (one or more) silk proteins are capable of forming a tertiary structure comprising a coiled-coil structure.

In one embodiment, the silk proteins are concentrated by: a) reducing the amount of surfactant in solution by adding a compound that precipitates the surfactant, and b) separating the solution comprising the silk proteins from the precipitate formed in step (a) to produce the silk dope.

Compounds that can be used to precipitate surfactants are known in the art and include a salt or carbohydrate; or a combination of two or more of these. Preferably, the salt is a potassium salt or a sodium salt. In one embodiment, the carbohydrate is a-cyclodextrin.

In another embodiment, silk proteins are concentrated by filtration, more preferably membrane filtration and, even more preferably, tangential flow filtration.

In one embodiment, the method further comprises increasing the concentration of silk proteins in the silk dope. This can be achieved by any method known in the art.

For example, the silk dope is dialyzed against a dehydration solution such as, for example, a solution comprising a hygroscopic polymer. Examples of hygroscopic polymers include, without limitation, polyethylene glycol, amylase and sericin, as well as a combination of two or more of these.

In a preferred embodiment, the silk dope comprises at least about 0.5% w / v silk proteins. In an additional embodiment, the silk dope comprises about 0.5% to about 15% silk proteins.

The cell can be any type of cell, typically a recombinant cell comprising an exogenous polynucleotide (s) that encodes, and capable of producing, the silk protein (s). Examples include, without limitation, bacterial cells, yeast cells, insect cells, plant cells or animal cells, or a combination of two or more of these. In a preferred embodiment, the cell is a bacterial cell. In a particularly preferred embodiment, the bacterial cell is Escherichia coli.

In a preferred embodiment, step (i) further comprises isolating inclusion bodies from the lysed cells.

The method can also comprise culturing the cells before step (i).

In a preferred embodiment, the portion of the silk protein that is capable of forming a tertiary structure that comprises a coiled-coil structure that comprises at least 10 copies of the heptad abcdefg sequence, and in which at least 25% of the amino acids in the a and d positions are alanine residues. More preferably, at least 25% of the amino acids at positions a, d and e are alanine residues.

In a further preferred embodiment, the silk protein comprises, most preferably it basically consists of, even more preferably it consists of, a sequence selected from: a) an amino acid sequence as provided in any of the IDS. SEQ. Nos 1 to 8, 17 to 24, 33 to 40, 49 to 56, 65 to 72, 81 to 88, 97 or 98, b) an amino acid sequence that is at least 30% identical to any one or more of the more than IDS. SEQ. Nos. 1 to 8, 17 to 24, 33 to 40, 49 to 56, 65 to 72, 81 to 88, 97 or 98, and c) a biologically active fragment of (a) or (b).

In the above aspect, it is preferred that as few silk proteins as possible are secreted by the cell. Consequently, it is preferred that silk proteins do not comprise an N-terminal signal sequence. Examples of silk proteins particularly useful for the above aspect include, without limitation, silk proteins that comprise, more preferably, which basically consist of and, furthermore, more preferably, they consist of, a sequence selected from: a) an amino acid sequence as provided in any of the IDS. SEQ. N ° s 1, 3, 5, 7, 17, 19, 21, 23, 33, 35, 37, 39, 49, 51, 53, 55, 65, 67, 69, 71, 81, 83, 85, 87 or 97, 5 b) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos 1, 3, 5, 7, 17, 19, 21, 23, 33, 35, 37, 39, 49, 51, 53, 55, 65, 67, 69, 71, 81, 35 83, 85, 87 or 97, and c) a biologically active fragment of (a) or (b).

In one embodiment, the silk proteins can be several of the same silk protein or a combination of two or more different silk proteins. In a preferred embodiment, if different silk proteins are used, there are four different silk proteins.

In a further embodiment, the silk proteins comprise a first silk protein which comprises, most preferably it basically consists of, even more preferably it consists of: a) an amino acid sequence as provided in any one of the IDS. SEQ. Nos. 1, 2, 17, 18, 33, 34, 49, 50, 65, 66, 81 or 82; b) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 1, 2, 17, 18, 33, 34, 49, 50, 65, 66, 81 or 82; and c) a biologically active fragment of (a) or (b), a second silk protein comprising, most preferably it basically consists of, even more preferably it consists of: d) an amino acid sequence as provided in any one of the IDS. SEQ. Nos. 3, 4, 19, 20, 35, 36, 51, 52, 67, 68, 83 or 84; e) an amino acid sequence that is at least 30% identical for any one or more of the IDS. SEQ. Nos. 3, 4, 19, 20, 35, 36, 51, 52, 67, 68, 83 or 84; and 5 f) a biologically active fragment of (c) or (d), a third silk protein comprising, more preferably basically consisting of, even more preferably consisting of: g) an amino acid sequence as provided in any of the 10 IDS. SEQ. Nos. 5, 6, 21, 22, 37, 38, 53, 54, 69, 70, 85 or 86; h) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 5, 6, 21, 22, 37, 38, 53, 54, 69, 70, 85 or 86; and i) a biologically active fragment of (g) or (h), and / or a fourth silk protein comprising, most preferably it basically consists of, even more preferably it consists of: 20 j) an amino acid sequence as provided in any of the IDS. SEQ. Nos. 7, 8, 23, 24, 39, 40, 55, 56, 71, 72, 87 or 88; k) an amino acid sequence that is at least 30% identical for any of the IDS. SEQ. Nos. 7, 8, 23, 25 24, 39, 40, 55, 56, 71, 72, 87 or 88; and 1) a biologically active fragment of (j) or (k). More preferably, in relation to the above aspect, the silk proteins comprise, or consist basically of, a first silk protein which comprises, more preferably consists basically of, even more preferably consists of: a) a sequence of amino acids as provided in any of the IDS. SEQ. Nos. 1, 17, 33, 49, 65 or 81; b) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 1, 17, 33, 49, 65 or 81; and c) a biologically active fragment of (a) or (b), a second silk protein comprising, most preferably it basically consists of, even more preferably it consists of: d) an amino acid sequence as provided in any of the IDS. SEQ. Nos. 3, 19, 35, 51, 67 or 83; e) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 3, 19, 35, 51, 67 or 83; and f) a biologically active fragment of (d) or (e), a third silk protein comprising, most preferably it basically consists of, even more preferably it consists of: g) an amino acid sequence as provided in any of the IDS. SEQ. Nos. 5, 21, 37, 53, 69 or 85; h) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 5, 21, 37, 53, 69 or 85; and i) a biologically active fragment of (g) or (h), and / or a fourth silk protein comprising, most preferably it basically consists of, even more preferably it consists of: any of the IDS. SEQ. Nos. 7, 23, 39, 55, 71 or 87; k) an amino acid sequence that is at least 30% identical to any one or more of the more than the IDS. SEQ. Nos. 7, 23, 39, 55, 71 or 87; and 1) a biologically active fragment of (j) or (k).

In one embodiment, the first silk protein, the second silk protein, the third silk protein and / or the fourth silk protein are produced by the same cells.

In an alternative embodiment, the first silk protein, the second silk protein, the third silk protein and / or the fourth silk protein are produced by different cells. In this embodiment, it is preferred that step (ii) comprises approximate equimolar amounts of the first silk protein, the second silk protein, the third silk protein and the fourth silk protein.

At any point up to and excluding step (iii), the silk proteins processed according to the invention can be prepared independently and combined. The silk proteins prepared separately can be the same or different. For example, a first silk protein as defined herein is expressed in a first cell and processed as defined in steps (i) and (ii), a second silk protein as defined herein is expressed in a second cell and processed as defined in steps (i) and (ii), and then the two solutions combined before step (iii) is carried out.

The surfactant and the ionic liquid solubilize the precipitated protein and allow the silk protein to remain in solution, allowing, at the same time, the formation of a coiled-coil structure during later stages.

In a preferred embodiment, the surfactant is an anionic surfactant. Examples of anionic surfactants useful for the invention include, without limitation, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate and other alkyl sulfate salts, sodium 1-octane sulfonate monohydrate, sodium lauryl sarcosinate, sodium lauryl sulfate ether (SLES), sodium taurodeoxycholate hydrate and alkyl benzene sulfonate; as well as a combination of two or more of these. In a preferred embodiment, the anionic surfactant is SDS.

In one embodiment, the ionic liquid comprises: i) an anion selected from chloride, bromide, iodide, thiocyanate, acetate, C1-C4-alkylsulfates, 15 methanesulfonates, tosylate, C1-C4-dialkylphosphates, hydrogen sulphate and tetrachloroaluminate, and ii) one selected cation of 1,3-CI ~ C4-dialkylimidazolium, 3-chloropyridinium, 4-dimethylaminopyridinium, 2-ethyl-4-aminopyridinium, 2-20 methylpyridinium, 2-ethylpyridinium, 2-ethyl-6-methylpyridinium, quinolinium, isoquinolinio, pyridinium, 1-C1- C4-alkylimidazolium, 1-methylimidazolium, 1,2-dimethylimidazolium, 1-n-butyl-imidazolium, 1,4,5-trimethylimidazolium, 1,4-dimethylimidazolium, imidazolium, 2-methylimidazolium, 1- butyl-25 2-methylimidazolium, 4 methylimidazolium, 1- (2'F-aminoethyl) imidazolium, 1-vinylimidazolium, 2-ethylimidazolium and benzotriazolium.

In a further preferred embodiment, the method yields at least about 0.1 g, more preferably at least 30 about 1 g, more preferably at least about 1.5 g, more preferably at least about 2 g, even more preferably at least about 2.5 g of silk protein (s) per liter of cultured cells.

In another aspect, the present invention provides a method for producing silk dope, the method comprising: i) obtaining cell culture supernatant, or a cellless expression system, producing one or more silk proteins, Ii) solubilization of silk proteins by their contact with a surfactant or an ionic liquid, and iii) concentration of silk proteins to produce the silk dope, in which (one or more) silk proteins are capable of forming 15 a tertiary structure comprising a coiled-coil structure.

In this respect, instead of the silk proteins in the cell being used to produce the silk dope, silk proteins are used that are secreted by the cells. As those skilled in the art will realize, step (i) of the first aspect and step (i) of the above aspect can be performed simultaneously or sequentially. In addition, at any corresponding stage, silk proteins derived from the cell and the supernatant could be combined and then processed together. For example, step (ii) of the first aspect and step (ii) of the above aspect can be performed separately and the silk proteins combined for further processing, including steps (iii) and (iv).

In a particularly preferred embodiment of the above aspect, step (i) further comprises increasing the concentration of silk proteins in the supernatant. This can be achieved by any method known in the art, for example, by contacting the supernatant with an agent that precipitates silk proteins, such as, without limitation, ammonium sulfate, trichloroacetic acid, perchloric acid and acetone.

In relation to the above aspect, it is preferred that as much of the silk proteins as possible is secreted by the cell. Consequently, it is preferred that silk proteins comprise an N-terminal signal sequence. Examples of silk proteins particularly useful for the above aspect include, without limitation, silk proteins that comprise, more preferably, that consist basically of and, even more so. preferably, which consist of, a selected sequence of: a) a sequence of amino acids as provided in any of the IDS. SEQ. Nos 2, 4, 6, 8, 18, 20, 22, 24, 34, 36, 38, 40, 50, 52, 54, 56, 66, 68, 70, 72, 82, 84, 86, 88 or 98 , b) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos 2, 4, 6, 8, 18, 20, 22, 24, 34, 36, 38, 40, 50, 52, 54, 56, 66, 68, 70, 72, 82, 84, 86, 88 or 98 , and c) a biologically active fragment of (a) or (b).

In a further embodiment, the silk proteins comprise a first silk protein which comprises, more preferably it basically consists of, even more preferably it consists of: any of the IDS. SEQ. Nos. 2, 18, 34, 50, 66 or 82; b) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 2, 18, 34, 50, 66 or 82; and c) a biologically active fragment of (a) or (b), a second silk protein comprising, most preferably it basically consists of, even more preferably it consists of: d) an amino acid sequence as provided in any of the IDS. SEQ. Nos. 4, 20, 36, 52, 68 or 84; e) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 4, 20, 36, 52, 68 or 84; and f) a biologically active fragment of (c) or (d), a third silk protein comprising, most preferably it basically consists of, even more preferably it consists of: g) an amino acid sequence as provided in any of the IDS. SEQ. Nos. 6, 22, 38, 54, 70 or 86; h) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 6, 22, 38, 54, 70 or 86; and i) a biologically active fragment of (g) or (h), and / or a fourth silk protein comprising, most preferably it basically consists of, even more preferably it consists of: j) an amino acid sequence as provided in any of the IDS. SEQ. Nos. 8, 24, 40, 56, 72 or 88; k) an amino acid sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos. 8, 24, 40, 56, 72 or 88; and l) a biologically active fragment of (j) or (k).

In a further aspect, the present invention provides a method for producing a silk fiber, the method comprising: extruding and / or extracting the silk dope produced by a method of the invention.

In one embodiment, the extrusion comprises the passage of the silk dope through a capillary tube of about 5 μm to about 500 μm.

In a particularly preferred embodiment, the method comprises: i) lysis of cells that produce one or more silk proteins and the isolation of inclusion bodies by the cells, ii) solubilization of silk proteins in the inclusion bodies by their contact with a surfactant or an ionic liquid, iii) concentration of silk proteins to produce silk dope, iv) increase in the concentration of silk proteins in the silk dope up to about 2% to about 10% by weight (%) of proteins silk, more preferably about 3% to about 6% by weight (%) of silk proteins, and vi) extrusion of the silk dope in a dehydration solution.

In relation to the above embodiment, the dehydration solution preferably comprises an alcohol such as, for example, methanol or ethanol, or a high concentration of salt such as, for example, MgCl2 or NaCl. Extrusion of silk fibers under these conditions is generally known in the art as wet spinning.

Preferably, the alcohol is methanol and the concentration of methanol in the solution is about 40% to about 80% v / v, more preferably about 50% to about 70% v / v. In this embodiment, the silk dope may comprise a single type of silk polypeptide, as defined herein, or two or more different types, for example, four different types.

In another aspect, the present invention provides a method for producing a silk film, wherein the method comprises molding the silk dope produced by a method of the invention.

In another aspect, the present invention provides silk dope produced by a method of the invention.

In a further aspect, the present invention provides a silk fiber produced by a method of the invention. Also provided is a silk film produced by a method of the invention.

In yet another aspect, the present invention provides a product that comprises a silk fiber and / or silk film of the invention.

As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.

Throughout this specification, the word "understand", or variations such as "comprise" or "understand", will be understood to imply the inclusion of an element, whole number or established step, or group of elements, whole numbers or steps, but not the exclusion of any other element, whole number or step, or group of elements, whole numbers or steps.

The following invention will be described by means of the following non-limiting examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ANNEXED DRAWINGS Figure 1. SDS-PAGE of purified inclusion bodies solubilized in SDS. The lanes correspond to the recombinant AmelFl-4 proteins; the scale is protein weight in kDa. Figure 2. Fourier auto-evolution of the amide I and II regions of the infrared spectra of native bee silk (A) and recombinant bee silk (B). The band distributions for structures are found in Table 2. Figure 3. Cross polarized microscopy of recombinant bee silk fibers (A) extracted in the air, and (B) extracted in the air and then extracted a second time in methanol. Figure 4. Cross polarized microscopy of recombinant bee silk fibers (A) extruded in a methanol bath, and (B) air-dried and then extracted a second time in a methanol bath up to x2 the length, or (C) dried in air and then extracted a second time in a methanol bath up to x4 length.

FUNDAMENTALS FOR SEQUENCE LISTING ID. SEQ. N °: 1 - Bee silk protein here called Xenospiral (also here called AmelFl) (less signal peptide). ID. SEQ. N °: 2 - Bee silk protein here called Xenospiral. ID. SEQ. N °: 3 - Bee silk protein here called Xenospira2 (also here called AmelF2) (less signal peptide). ID. SEQ. N °: 4 - Bee silk protein here called Xenospira2. ID. SEQ. N °: 5 - Bee silk protein here called Xenospira3 (also here called AmelF3) (less signal peptide). ID. SEQ. N °: 6 - Bee silk protein here called Xenospira3. ID. SEQ. N °: 7 - Bee silk protein here called Xenospira4 (also here called AmelF4) (less signal peptide). ID. SEQ. N °: 8 - Bee silk protein here called Xenospira4. ID. SEQ. N °: 9 - Nucleotide sequence that encodes Xenospiral bee silk protein (less region that encodes the signaling peptide). ID. SEQ. N °: 10 - Nucleotide sequence that encodes Xenospiral bee silk protein. ID. SEQ. N °: 11 - Nucleotide sequence that encodes Xenospira2 bee silk protein (less region that encodes the signaling peptide). ID. SEQ. N °: 12 - Nucleotide sequence that encodes Xenospira bee silk protein2. ID. SEQ. N °: 13 - Nucleotide sequence that encodes Xenospira3 bee silk protein (less region that encodes the signaling peptide). ID. SEQ. N °: 14 - Nucleotide sequence that encodes bee silk protein Xenospira3. ID. SEQ. N °: 15 - Nucleotide sequence that encodes Xenospira4 bee silk protein (less region that encodes the signaling peptide). ID. SEQ. N °: 16 - Nucleotide sequence that encodes Xenospira bee silk protein4. ID. SEQ. N °: 17 - Wasp-silk silk protein here called BBF1 (less signal peptide). ID. SEQ. N °: 18 - Wasp silk protein from mangangá called BBF1. ID. SEQ. N °: 19 - Wasp silk protein from mangangá here called BBF2 (less signal peptide). ID. SEQ. N °: 20 - Wasp silk protein from mangangá called BBF2. ID. SEQ. N °: 21 - Wasp-silk silk protein here called BBF3 (less signal peptide). ID. SEQ. N °: 22 - Wasp silk protein from mangangá called here BBF3. ID. SEQ. N °: 23 - Wasp silk protein from mangangá here called BBF4 (less signal peptide). ID. SEQ. N °: 24 - Wasp silk protein from mangangá called here BBF4. ID. SEQ. N °: 25 - Sequence of nucleotides that encodes silk protein of wasp of mangangá mangangá BBF1 (less region that encodes the signal peptide). ID. SEQ. N °: 26 - Nucleotide sequence encoding BBF1 silk wasp silk protein. ID. SEQ. N °: 27 - Nucleotide sequence encoding BBF2 silk wasp protein (less region encoding signaling peptide). encodes BBF2 silk wasp silk protein. ID. SEQ. N °: 29 - Nucleotide sequence encoding BBF3 silk wasp silk protein (less region encoding signaling peptide). ID. SEQ. N °: 30 - Nucleotide sequence encoding BBF3 silk wasp silk protein. ID. SEQ. N °: 31 - Nucleotide sequence encoding BBF4 silk wasp silk protein (less region encoding signaling peptide). ID. SEQ. N °: 32 - Nucleotide sequence that encodes BBF4 silk wasp silk protein. ID. SEQ. N °: 33 - Bulldog ant silk protein here called BAF1 (less signal peptide). ID. SEQ. N °: 34 - Bulldog ant silk protein here called BAF1. ID. SEQ. N °: 35 - Bulldog ant silk protein here called BAF2 (less signal peptide). ID. SEQ. N °: 36 - Bulldog ant silk protein here called BAF2. ID. SEQ. N °: 37 - Bulldog ant silk protein here called BAF3 (less signal peptide). ID. SEQ. N °: 38 - Bulldog ant silk protein here called BAF3. ID. SEQ. N °: 39 - Bulldog ant silk protein here called BAF4 (less signal peptide). ID. SEQ. N °: 40 - Bulldog ant silk protein here called BAF4. ID. SEQ. N °: 41 - Nucleotide sequence encoding BAF1 ant-bulky silk protein (less region encoding signaling peptide). ID. SEQ. N °: 42 - Nucleotide sequence encoding BAF1 ant-bulky silk protein. ID. SEQ. N °: 43 - Sequence of nucleotides encoding BAF2 ant-bulky silk protein (less region encoding signaling peptide). ID. SEQ. N °: 44 - Sequence of nucleotides encoding BAF2 ant-bulky silk protein. ID. SEQ. N °: 45 - Nucleotide sequence encoding BAF3 ant-bulky silk protein (minus 30 region encoding the signal peptide). ID. SEQ. N °: 46 - Nucleotide sequence encoding BAF3 ant-bulky silk protein. ID. SEQ. N °: 47 - Nucleotide sequence encoding BAF4 ant-bulky silk protein (less region encoding signaling peptide). ID. SEQ. N °: 48 - Nucleotide sequence encoding BAF4 ant-bulky silk protein. ID. SEQ. N °: 49 - Ant-weaver silk protein here called GAF1 (less signal peptide). ID. SEQ. N °: 50 - Ant-weaver silk protein here called GAF1. ID. SEQ. N °: 51 - Ant-weaver silk protein here called GAF2 (less signal peptide). ID. SEQ. N °: 52 - Ant-weaver silk protein here called GAF2. ID. SEQ. N °: 53 - Ant-weaver silk protein here called GAF3 (less signal peptide). ID. SEQ. N °: 54 - Ant-weaver silk protein here called GAF3. weaver here called GAF4 (less signal peptide). ID. SEQ. N °: 56 - Ant-weaver silk protein here called GAF4. ID. SEQ. N °: 57 - Nucleotide sequence encoding weaver ant silk protein GAF1 (less region encoding signaling peptide). ID. SEQ. N °: 58 - Nucleotide sequence encoding weaver ant silk protein GAF1. ID. SEQ. N °: 59 - Nucleotide sequence encoding weaver ant silk protein GAF2 (less region encoding signaling peptide). ID. SEQ. N °: 60 - Nucleotide sequence encoding weaver ant silk protein GAF2. ID. SEQ. N °: 61 - Sequence of nucleotides that encodes silk ant-weaver protein GAF3 (less region that encodes the signal peptide). 15 ID. SEQ. N °: 62 - Nucleotide sequence that encodes silk ant-weaver protein GAF3. ID. SEQ. N °: 63 - Sequence of nucleotides that encodes silk ant-weaver protein GAF4 (less region that encodes the signal peptide). ID. SEQ. N °: 64 - Nucleotide sequence encoding weaver ant silk protein GAF4. ID. SEQ. N °: 65 - Wasp silk protein here called Vssilk3 (less signal peptide). ID. SEQ. N °: 66 - Wasp silk protein here called Vssilk3. ID. SEQ. N °: 67 - Wasp silk protein here called Vssilk4 (less signal peptide). called Vssilkí. ID. SEQ. N °: 69 - Wasp silk protein here called Vssilk2 (less signal peptide). ID. SEQ. N °: 70 - Wasp silk protein here called Vssilk2. ID. SEQ. N °: 71 - Wasp silk protein here called Vssilkl (less signal peptide). ID. SEQ. N °: 72 - Wasp silk protein here called Vssilkl. ID. SEQ. N °: 73 - Nucleotide sequence that encodes wasp silk protein Vssilk3 (less region that encodes the signal peptide). ID. SEQ. N °: 74 - Nucleotide sequence encoding Vssilk3 wasp silk protein. ID. SEQ. N °: 75 - Nucleotide sequence that encodes Vssilkl wasp silk protein (less region that encodes the signal peptide). ID. SEQ. N °: 76 - Nucleotide sequence encoding Vssilkl wasp silk protein. ID. SEQ. N °: 77 - Nucleotide sequence that encodes Vssilk2 wasp silk protein (less region that encodes the signal peptide). ID. SEQ. N °: 78 - Nucleotide sequence encoding Vssilk2 wasp silk protein. ID. SEQ. N °: 79 - Nucleotide sequence that encodes Vssilkl wasp silk protein (less region that encodes the signaling peptide). ID. SEQ. N °: 80 - Nucleotide sequence encoding Vssilkl wasp silk protein. Asian protein called silk protein 1 (also called ABS1) (less signal peptide). ID. SEQ. N °: 82 - Asian bee silk protein called silk protein 1 (also called ABS1). ID. SEQ. N °: 83 - Asian bee silk protein called silk protein 2 (also called ABS2) (less signal peptide). ID. SEQ. N °: 84 - Asian bee silk protein called silk protein 2 (also called ABS2). ID. SEQ. N °: 85 - Asian bee silk protein called silk protein 3 (also called ABS3) (less signal peptide). ID. SEQ. N °: 86 - Asian bee silk protein called silk protein 3 (also called ABS3). ID. SEQ. N °: 87 - Asian bee silk protein called silk protein 4 (also called ABS4) (less signal peptide). ID. SEQ. N °: 88 - Asian bee silk protein called silk protein 4 (also called ABS4). ID. SEQ. N °: 89 - Nucleotide sequence that encodes ABS1 Asian bee silk protein (less region that encodes the signal peptide). ID. SEQ. N °: 90 - Nucleotide sequence encoding ABS1 Asian bee silk protein. ID. SEQ. N °: 91 - Sequence of nucleotides (region that encodes the signal peptide). ID. SEQ. N °: 92 - Nucleotide sequence encoding ABS2 Asian bee silk protein. ID. SEQ. N °: 93 - Nucleotide sequence that encodes ABS3 Asian bee silk protein (less region that encodes the signal peptide). ID. SEQ. N °: 94 - Nucleotide sequence that encodes ABS3 Asian bee silk protein. ID. SEQ. N °: 95 - Nucleotide sequence that encodes ABS4 Asian bee silk protein (less region that encodes the signal peptide). ID. SEQ. N °: 96 - Nucleotide sequence encoding ABS4 Asian bee silk protein. ID. SEQ. N °: 97 - Chrysopid silk protein here called MalFl (less signal peptide). ID. SEQ. N °: 98 - Chrysopid silk protein here called MalFl. ID. SEQ. N °: 99 - Nucleotide sequence that encodes MalFl crispy silk protein (less region that encodes the signal peptide). ID. SEQ. N °: 100 - Nucleotide sequence that encodes MalFl crispy silk protein. IDS. SEQ. Nos 101 to 108 - Oligonucleotide primers.

DETAILED DESCRIPTION OF THE INVENTION General techniques and definitions

Unless specifically defined differently, all technical and scientific terms used herein should be considered to have the same meaning commonly understood by those skilled in the art (for example, in cell culture, molecular genetics, silk processing, immunology, immunohistochemistry, protein chemistry and biochemistry).

Unless otherwise indicated, recombinant protein, cell culture and immunological techniques used in the present invention are standard procedures, well known to those skilled in the art. These techniques are described and explained in the literature in sources such as, for example, J. Perbal, "A Practical Guide to Molecular Cloning", John Wiley and Sons (1984), J. Sambrook et al., "Molecular Cloning: A Laboratory Manual ", Cold Spring Harbor Laboratory Press (1989), TA Brown (editor)," Essential Molecular Biology: A Practical Approach ", Volumes 1 and 2, IRL Press (1991), DM Glover and B.D. Hames (editors), "DNA Cloning: A Practical Approach", Volumes 1-4, IRL Press (1995 and 1996) and F.M. Ausubel et al. (editors), "Current Protocols in Molecular Biology", Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (editors) "Current Protocols in Immunology", John Wiley & Sons (including all updates to date).

The term "and / or", for example, "X and / or Y" should be understood to mean "X and Y" or "X or Y", and should be taken to provide explicit support for both meanings or for one of meanings.

As used herein, the terms "silk protein" and "silk polypeptide" refer to a fibrous protein / polypeptide that can be used to produce a silk fiber, and / or a fibrous protein complex.

As used herein, the term "one or more silk proteins" refers to the process that possibly uses two or more different types of silk proteins such as, for example, a first silk protein, second silk protein etc., as herein defined. Thus, in this context, a silk protein means a population of identical silk protein molecules sufficient to produce silk dope.

As used herein, the term "capable of forming a tertiary structure comprising a coiled-coil structure" refers to the ability of proteins to form said structures under suitable conditions. For example, when processed to produce silk fibers, proteins form said structures. Furthermore, this term does not mean that the whole protein is capable of forming a coiled-coil structure, only a portion of it. In one embodiment, about 45% to about 90%, more preferably about 55% to about 70%, and even more preferably, about 60% to about 66%, of the silk protein is able to form a tertiary structure comprising a coiled-coil structure.

As used herein, the term "silk dope" refers to an aqueous solution that comprises silk proteins. Preferably, the silk dope comprises at least 0.05% w / v, more preferably at least 0.1% w / v, even more preferably, at least 0.5% w / v, of a silk protein, as defined herein. In one embodiment, the silk dope produced by a method of the invention comprises about 0.5% to about 15% (% by weight) of silk protein. However, if the subsequent step of increasing the silk protein concentration in the silk dope is not carried out, the most typical yield is about 0.5% to about 4% (weight%) of silk protein. The silk dope produced using a method of the invention is capable of extrusion for the formation of a fiber and / or film molding.

As used herein, a "silk fiber" refers to filaments that comprise silk proteins that can be braided into various items, for example, textiles.

As used herein, the term "reduction of the amount of surfactant solution", or variations thereof which include a reduction in the amount of ionic liquid, means that the total amount of surfactant or ionic liquid is decreased. In one embodiment, the concentration of surfactant or ionic liquid after reduction is less than about 10 mM, more preferably less than 5 mM, and even more preferably less than 1 mM, before any step involving additional concentration of the solution.

As used herein, the "dehydration solution" is any solution, preferably an aqueous solution, which has a lower water concentration in solution than the silk dope that is to be concentrated.

As used herein, the term "solubilization", when referring to silk proteins that are being brought into contact with a surfactant or ionic liquid, means that the surfactant or ionic liquid associates with silk proteins and keeps them in solution throughout avoid their aggregation. This is in contrast to the silk proteins observed SB in cells, especially in inclusion bodies.

The term "signal peptide" or "N-terminal signal sequence" and variations thereof refer to an amino terminal protein / peptide that precedes a secreted mature protein. The signal peptide is cleaved from it and therefore is not present in the mature protein. Signaling peptides have the function of directing and transporting secreted proteins across cell membranes. The signal peptide is also called a signal sequence, and is well known in the art.

Coiled-coil silk proteins The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain that may or may not be modified by adding groups of non-amino acids. The terms "proteins" and "polypeptides", as used herein, also include variants, mutants, modifications, analogs and / or derivatives of the silk proteins described herein. In a preferred embodiment, a silk protein used in the invention is composed only of naturally occurring amino acids. The 0% identity of a protein is determined by GAP analysis (Needleman and Wunsch, 1970) (GCG program) with a gap creation penalty = 5 and a gap extension penalty = 0.3. The searched sequence is at least 15 amino acids long, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the sequence searched is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the sequence searched is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the sequence searched is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over the entire length.

As used herein, a "biologically active" fragment is a portion of a protein of the invention that maintains a defined activity of the protein in full length, specifically the ability to be used to produce silk. Biologically active fragments can be of any size, as long as they maintain the defined activity. The term "which basically consists of", or variations thereof, means that the defined amino acid sequence may have few, for example, one, two, three or four, additional amino acids, compared to that defined. For example, when absent from the defined sequence, an N-terminal methionine can be added. The term "consists of", or variations thereof, means that the defined sequence has no additional amino acids or has fewer amino acids when compared to the defined sequence, particularly at the N and C terminals.

With respect to a defined protein, it will be noted that values greater than% of identity than those provided above will encompass preferred modalities. Thus, when applicable, in light of the minimum values of% identity, it is preferred that the protein comprises an amino acid sequence that is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, most preferably at least 80%, more preferably at least 85%, more preferably at least less 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferable at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, most preferably at least 99.8% and even more preferably at least least 99.9% identical to the ID No. SEQ. relevant.

Amino acid sequence mutants of naturally occurring silk proteins described herein can be prepared by introducing appropriate nucleotide changes in a nucleic acid encoding the silk protein, or by in vitro synthesis of the desired protein. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be done to arrive at the final construction, as long as the final protein product has the desired characteristics.

Mutant (altered) proteins can be prepared using any methodology known in the art. For example, a polynucleotide of the invention can undergo mutagenesis in vitro. These in vitro mutagenesis techniques include subcloning the polynucleotide into a suitable vector, transforming the vector into a "mutant" strain such as E. coli XL-1 red (Stratagene), and the propagation of bacteria transformed by a adequate number of generations. In another example, the polynucleotides of the invention are subjected to DNA shuffling techniques, as widely described by Harayama (1998). Such DNA shuffling techniques may include genes of the invention, possibly in addition to genes related to those of the present invention, for example, silk genes from Himenoptera or Neuroptean species other than the specific species characterized here. Products derived from mutated / altered DNA can easily be evaluated using the techniques described here to determine whether they can be used as silk proteins.

In the design of amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the characteristic (s) to be modified. The mutation sites can be modified individually or in series, for example, by (1) substituting first with conservative amino acid choices and then with more radical selections, depending on the results obtained, (2) deletion of the target residue, or (3 ) insertion of other waste adjacent to the sites located.

Deletions or insertions of amino acid sequences generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and, typically, about 1 to 5 contiguous residues.

Substitution mutants have at least one amino acid residue 5 in the protein molecule removed and a different residue inserted in its place. Sites of greatest interest for substitution mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues 10 obtained from various strains or species are identical. These positions can be important for biological activity. These sites, especially those that fall within a sequence of at least three other identically preserved sites, are preferably replaced in a relatively conservative manner. These conservative substitutions are shown in Table 1 under the heading "exemplary substitutions". Table 1. Exemplary substitutions Residue original Exemplary substitutions

Coiled-coil structures of silk proteins are characterized by heptad repetitions represented by the consensus sequence (abode fg). In a preferred embodiment, the portion of the protein that has a coiled-coil structure comprises at least 10 copies of the heptad abcdefg sequence, and at least 25% of the amino acids at positions a and d are alanine residues.

In a preferred embodiment, the protein having a coiled-coil structure comprises at least 12 consecutive copies, more preferably at least 15 consecutive copies and, even more preferably, at least 18 consecutive copies of the heptad. In additional embodiments, the protein that has a coiled-coil structure can have up to at least 28 copies of the heptad. Typically, copies of the 15 heptad will be repeated in tandem. However, they do not necessarily have to be perfect tandem repeats, for example, as shown in Figures 5 and 6 of WO 2007/038837, few amino acids can be found between two heptads, or few truncated heptads can be found (see, for example , Xenospiral in Figure 5 of WO 2007/038837).

Guidelines regarding amino acid substitutions that can be made to silk proteins that have a coiled-coil structure are provided in Figures 5 and 6, as well as in Tables 6 to 10, of WO 2007/038837. When an amino acid substitution envisaged as useful on the basis of the experimental data provided herein is in some conflict with the exemplary substitutions provided in Table 1 of WO 2007/038837, it is preferred that the substitution based on the experimental data be used.

The coiled-coil structures of silk proteins have a high content of alanine residues, particularly at the amino acid positions a, d and e of heptad. However, positions b, c, f and g also have a high frequency of alanine residues. In a preferred embodiment, at least 15% of the amino acids at positions a, / or and of heptads are residues of alanine, more preferably at least 25%, more preferably at least 30%, more preferably at least 40% and, even more preferably at least 50%. In a further preferred embodiment, at least 25% of the amino acids at both the a and d positions of the heptads are residues of alanine, more preferably at least 30%, more preferably at least 40% and, even more preferably, at least 50%. In addition, it is preferred that at least 15% of the amino acids at the b, c, f and g positions of the heptads are residues of alanine, more preferably at least 20% and, even more preferably, at least 25%.

Typically, heptads will not comprise any proline or histidine residues. In addition, heptads will comprise few (1 or 2), if any, residues of phenylalanine, methionine, tyrosine, cysteine, glycine or tryptophan. In addition to alanine, common amino acids (for example, more than 5%, more preferably more than 10%) in heptads include leucine (particularly at bed positions), serine (particularly at positions b, eef), glutamic acid (particularly at positions c , eef), lysine (particularly at positions b, c, d, f and g), as well as arginine at position g.

In a preferred embodiment, heptads are determined using the MARCOIL pattern recognition program (Delorenzi and Speed, 2002).

Proteins (and polynucleotides) useful for the methods of the invention can be purified (isolated) from a wide variety of Hymenoptera and Neuropteran species. Examples of Hymenoptera include, without limitation, any species of the Suborder Apocrita (bees, ants and wasps), which include the following insect families: Chrysididae (wasp-cuckoo), Formicidae (ants), Mutillidae (velvet ants), Pompilidae ( spider wasps), Scoliidae, wasp (paper wasps, pot wasps, wasps), Agaonidae (fig wasps), Chalcididae (chalcidids), Eucharitidae (eucharitids), Eupelmidae (eupelmids), pteromalidae (pteromalidae), Pteromalidae Evaniidae (wasps), Braconidae, Ichneumonidae (ichneumons), Megachilidae, Apidae, Colletidae, Halictidae and Melittidae (oil-collecting bees). Examples of Neuropterans include species from the following insect families: Mantispidae, Chrysopidae (crisopideos), Myrmeleontidae (antlions) and Ascalaphidae (.owlflies). These additional proteins (and polynucleotides) can be characterized using the same procedures described here for silks from Bombus terrestris, Myrmecia forficata, Oecophylla smaragdina and Mallada signata.

Also included within the scope of the invention are proteins that are differentially modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, binding to a antibody molecule or other cell ligand etc. These modifications can serve to increase the stability and / or bioactivity of the protein.

Polynucleotides The term "polynucleotide" is used interchangeably with the term "nucleic acid" here. The term "exogenous", in the context of a polynucleotide, refers to the polynucleotide when present in a cell, or in a cellless expression system, in an altered amount, compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell can be a cell that comprises a non-endogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded protein. An exogenous polynucleotide of the invention includes polynucleotides that have been separated from other components of the transgenic (recombinant) cell, or cellless expression system, in which it is present, and polynucleotides produced in those cells or cellless systems that are subsequently purified from at least some other components. The% identity of a polynucleotide is determined by GAP analysis (Needleman and Wunsch, 1970) (GCG program) with a gap creation penalty = 5, and a gap extension penalty = 0.3. Unless defined differently, the sequence searched is at least 45 nucleotides in length, and GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the searched sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the sequence searched is at least 300 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over the entire length.

With respect to the defined polynucleotides, it will be observed that values of% identity greater than those provided above will encompass preferred modalities. Thus, when applicable, in light of the minimum values of% identity, it is preferred that a polynucleotide of the invention comprises a sequence that is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, most preferably at least 80%, more preferably at least 85%, more preferably at least less 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferable substantially at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, most preferably at least 99.8% and, even more preferably, at least least 99.9% identical to the ID No. SEQ. relevant.

In one embodiment, a polynucleotide encoding a silk protein useful for the invention comprises, most preferably it basically consists of, even more preferably it consists of, a sequence selected from: a) a sequence of nucleotides as provided in any of the IDS. SEQ. In 9 to 16, 25 to 32, 41 to 48, 57 to 64, 73 to 80, 89 to 96, 99 or 100, b) a nucleotide sequence that is at least 30% identical to any one or more of the IDS. SEQ. Nos 9 to 16, 25 to 32, 41 to 48, 57 to 64, 73 to 80, 89 to 96, 99 or 100, and c) a biologically active fragment encoding a portion of (a) or (b).

When it is preferred that the smallest possible number of silk proteins be secreted by the cell, the encoded silk proteins do not comprise an N-terminal signal sequence. Examples of polynucleotides encoding these silk proteins include those that most preferably comprise basically in, even more preferably, which consist of, a selected sequence of: a) a sequence of nucleotides as provided in any of the IDS. SEQ. Nos 9, 11, 13, 15, 25, 27, 29, 31, 41, 43, 45, 47, 57, 59, 61, 63, 73, 75, 77, 79, 89, 91, 93, 95 or 97 , b) a sequence of nucleotides that is at least 30% identical to any one or more of the IDS. SEQ. Nos 9, 11, 13, 15, 25, 27, 29, 31, 41, 43, 45, 47, 57, 59, 61, 63, 73, 75, 77, 79, 10 89, 91, 93, 95 or 97, and c) a biologically active fragment of (a) or (b).

Other embodiments of the invention are based on the expression of silk proteins with an N-terminal signal sequence, and / or the co-production (in the same cells or in different cells) of a first silk protein, second silk protein, third protein and / or fourth silk protein, as defined herein. Based on the sequence information provided in the sequence listing, those skilled in the art could easily identify representative polynucleotides for expression for each embodiment of the invention.

Polynucleotides for use in the methods of the present invention may have, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions or substitutions of nucleotide residues. The mutants can be naturally occurring (that is, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis in the nucleic acid).

Polynucleotides for use in the invention can also hybridize to a nucleotide sequence that encodes a silk protein, as provided herein, for example, one or more of the IDS. SEQ. Nos 9 to 16, 25 to 32, 41 to 48, 57 to 64, 73 to 80, 89 to 96, 99 and 100, under strict conditions. The term "stringent hybridization conditions", and the like, as used herein, refers to the parameters with which the technique is familiar, including the variation of the oligonucleotide length hybridization temperature. Nucleic acid hybridization parameters can be found in references that compile these methods, Sambrook, et al. (supra), and Ausubel, et al. {supra). For example, stringent hybridization conditions, as used herein, may refer to hybridization at 65 ° C in hybridization buffer (3.5x SSC, Ficoll 0.02%, polyvinyl pyrrolidone 0.02%, bovine serum albumin 0.02 % (BSA), 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA), followed by one or more washes in 0.2x SSC, 0.01% BSA at 50 ° C.

Nucleic acid constructs Cells for use in the methods of the invention will typically comprise a nucleic acid construct that encodes the silk protein (s). The construct can be integrated into the cell's genome, or be extrachromosomal, for example, a recombinant vector. A vector of this type contains sequences of heterologous polynucleotides, that is, sequences of polynucleotides that are not found naturally adjacent to the polynucleotide molecule that encodes the silk protein, and that are preferably derived from a species other than the species from which it (s) polynucleotide molecule (s) is derived. The vector can be RNA or DNA, prokaryotic or eukaryotic, and is typically a transposon (for example, described in US 5,792,294), a virus or a plasmid.

One type of recombinant vector comprises a polynucleotide molecule that encodes the silk protein operably linked to an expression vector. The phrase "operably linked" refers to the insertion of a polynucleotide molecule into an expression vector such that the molecule is capable of being expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and effecting the expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be prokaryotic or eukaryotic, and are typically viruses or plasmids. The expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including bacterial, fungal, endoparasitic, arthropod, insect, animal and plant cells. Particularly preferred expression vectors of the present invention can direct gene expression in plant cells. The vectors of the invention can also be used to produce the protein in a cellless expression system, and these systems are well known in the art.

In particular, the nucleic acid construct contains regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of molecules polynucleotide. Transcription control sequences are sequences that control the initiation, elongation and termination of transcription. Particularly important transcription control sequences are those that control the initiation of transcription, such as promoter, enhancer, operator and repressor sequences. Suitable transcriptional control sequences include any transcriptional control sequence that can function in at least one of the recombinant cells of the present invention. Several of these transcription control sequences are known to those skilled in the art. Preferred transcriptional control sequences include those that function in bacterial, yeast, arthropod, plant or mammalian cells, such as, without limitation, tac, lac, trp, trc, oxy-pro, omp / lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, subgenomic alfavirus promoters (eg, subgenomic promoters of the Sindbis virus), antibiotic resistance gene, baculovirus , Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (e.g., intermediate early promoters), simio virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus , thermal shock, phosphate and nitrate transcription control sequences, in addition to other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells.

As presented above, one aspect of the invention is based on the fact that the silk protein is secreted by the cell, typically due to the presence of a N-terminal signal sequence. Examples of suitable signal segments include, without limitation, tissue plasminogen activator ( t-PA), interferon, interleukin, growth hormone, viral envelope glycoprotein signal segments, Nicotiana nectarin signal peptide (US 5,939,288), tobacco extensin signal, the oleosin oily body binding protein signal , basic vacuolar chitinase signal peptide from Arabidopsis thaliana, in addition to native signal sequences of the silk polypeptides defined here.

Cells Most methods of the invention are based on the use of cells that produce (one or more) silk proteins as defined herein. The transformation of a polynucleotide molecule into a cell can be achieved by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, without limitation, transfection, electroporation, microinjection, lipofection, adsorption and protoplast fusion. A recombinant cell can remain single-celled or it can grow into a multicellular tissue, organ or organism. The transformed polynucleotide molecules may remain extrachromosomal or may integrate into one or more sites within a chromosome of the transformed (ie, recombinant) cell in such a way that their ability to be expressed is retained.

Suitable host cells for transformation include any cell that can be transformed with a polynucleotide that encodes a silk polypeptide as defined herein. Host cells may be endogenously (i.e., naturally) capable of producing silk polypeptides or may be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule as defined herein. Host cells can be any cell capable of producing at least one silk protein as defined herein, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. Examples of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (hamster puppy kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS cells (for example, COS cells -7) and Vero cells. Additional examples of host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic myoblast cells from mouse G8 (eg, ATCC CRL 1246). Suitable additional mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (for example, human, murine, or chicken embryo cell lines), myeloma cell lines, ovarian cells from Chinese hamster, NIH / 3T3 mouse cells, LMTK cells and / or HeLa cells. Particularly preferred host cells are bacterial cells.

Those skilled in the art can easily determine suitable culture conditions, for example, media, temperature and time, for a particular cell type. For example, in one embodiment, Escherichia coli cells are grown at around 30 ° C to about 37 ° C for a period of about 24 hours to about 48 hours.

Recombinant DNA technologies can be used to increase the expression of a polynucleotide molecule transformed by manipulation, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resulting transcripts are translated and the efficiency of post-translation modifications.

Recombinant techniques useful for increasing expression of polynucleotide molecules include, without limitation, polynucleotide molecules operably linked to plasmids with high copy number, integration of the polynucleotide molecule into one or more chromosomes of the host cell, addition of sequences of stability of vector to plasmids, substitutions or modifications of transcriptional control signals (for example, promoters, operators, enhancers); substitutions or modifications of translation control signals (for example, ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules to match the use of codons in the host cell, and the deletion of sequences that destabilize transcripts.

Silk dope production The present invention relates to silk dope production methods, which can then be used for a wide variety of applications.

One step of an aspect of the invention is related to the lysis of cells to release silk proteins produced and contained within the cells. This step can be performed by any means known in the art. For example, the cell suspension is typically centrifuged to pellet the cells and resuspend cells in a more concentrated solution ready for lysis. The cells can be lysed, for example, by passages through a French press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternative methods for cell lysis, for example, bacterial cells, are well known to those skilled in the art (see, for example, Sambrook et al, supra). Various kits are available for cell lysis and are well known in the art, for example, the Bugbuster kit (Novagen) and the ProteaPrep kit (Protea Biosciences, Inc.). The present inventors have identified that silk proteins as defined herein expressed in bacteria were insoluble aggregates ("inclusion bodies"). In a preferred embodiment, the method includes isolating these inclusion bodies. Several protocols are suitable for purification of protein inclusion bodies. For example, the purification of inclusion bodies typically involves the extraction, separation and / or purification of inclusion bodies by rupture of bacterial cells by the methods discussed above. In one embodiment, the cells are lysed, the cell membranes solubilized, and the insoluble fraction that comprises the inclusion bodies is isolated for further processing.

One aspect of the invention is based on increasing the concentration of silk proteins in the supernatant. Again, this can be achieved by any method known in the art. In one embodiment, this is achieved by contacting the supernatant with an agent that precipitates silk proteins, such as, without limitation, ammonium sulfate, trichloroacetic acid, perchloric acid and acetone, or commercial precipitating cocktails such as PlusOne (Amersham Biosciences) or Perfect-Focus (Geno Technology Inc.).

An optional step of the invention for the production of silk dope, but nevertheless preferred in cases where the yield of silk protein is not high enough, comprises increasing the concentration of silk proteins in the silk dope. Again, this can be achieved by any method known in the art for increasing the concentration of a protein in an aqueous solution. In a particularly useful embodiment, the silk dope is concentrated by dialysis against a dehydration solution such as, for example, a solution comprising a hygroscopic polymer. Examples of suitable hygroscopic polymers include, without limitation, polyethylene glycol (PEG), amylase and sericin, or a combination of two or more of these. PEG molecules are available in several molecular sizes and the selection of PEG will be determined by the membrane chosen for dialysis and the required concentration rate. Preferably, the PEG has a molecular weight of about 8,000 to about 10,000 g / mol and has a concentration of about 25% to about 50%.

Surfactants In one embodiment, a silk dope production step as defined herein involves the use of a surfactant. The present inventors have surprisingly found that surfactants, for example, SDS, allow silk proteins, as defined herein, to remain in solution, while allowing the formation of a coiled-coil structure when the concentration of the surfactant is reduced.

In one embodiment, the surfactant is an anionic surfactant. Examples of anionic surfactants useful for the invention include, without limitation, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate and other alkyl sulfate salts, sodium octane sulfonate monohydrate 1, sodium lauroyl sarcosinate, sodium lauryl sulfate ( SLES), sodium taurodeoxycholate hydrate and alkyl benzene sulfonate; or a combination of two or more of these. In a preferred embodiment, the anionic surfactant is SDS.

Any concentration of the surfactant that increases the solubility of silk proteins can be used. For example, at least about 0.1% v / v of the surfactant is used. In one embodiment, about 0.1% to about 10% v / v, more preferably, about 0.5% to about 2% v / v or about 0.5% to about 5% v / v v of the surfactant is used.

A further step in the methods of the invention for producing silk dope comprises reducing the amount of surfactant in solution by adding a compound that precipitates the surfactant to aid in the correct folding of silk proteins. Any compound that combines and reduces the solubility of the surfactant can be used. Examples include, without limitation, a salt or carbohydrate such as, for example, a-cyclodextrin; or a combination of two or more of these. Preferably, the salt is a potassium salt or a sodium salt. Preferably, the potassium salt is potassium chloride and the sodium salt is sodium acetate. Any concentration of the compound that results in a reduction in the amount of surfactant in solution can be used. For example, the compound is added to a final concentration of about 1 mM to about 1 M, more preferably about 40 mM to about 100 mM, or about 40 mM to 400 mM.

A further step in the methods of the invention for producing silk dope comprises separating the solution from the precipitate formed after adding the compound. This can be achieved by any method known in the art, such as, for example, with the use of centrifugation, for example, at 16,000 g for 5 minutes, and removal of the supernatant (solution) that comprises (which is) the silk dope. Preferably, after that step, the silk proteins constitute at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97 %, more preferably at least 99% and, even more preferably, 100% of the protein in solution.

Ionic liquids Generally, ionic liquids can be defined as compounds that are composed entirely of ions and are liquid at temperatures of less than about 100 ° C, preferably less than about 85 ° C. As used in the present invention, ionic liquids generally comprise one or more anions and one or more cations. In preferred embodiments, ionic liquids comprise organic cations created by derivatization of one or more compounds to include substituents, for example, alkyl, alkenyl, alkynyl, alkoxy, alkenoxy, alkinoxy, various aromatics, for example, phenyl (substituted or unsubstituted) , benzyl (substituted or unsubstituted), phenoxy (substituted or unsubstituted) and benzoxy (substituted or unsubstituted), and several heterocyclic aromatics that have one, two or three hetero atoms in their ring portion, said heterocyclics being substituted or not replaced. Derivatized compounds include, without limitation, imidazoles, pyrazoles, thiazoles, isothiazoles, azathiozoles, oxothiazoles, oxazins, oxazolines, oxazaborols, dithiozoles, triazoles, delenozoles, oxafosfols, pyrroles, borols, furans, indophones, pentols, indoles, pentols, indoles, pentols, indoles, phosphenes, oxazoles, isoxazoles, isotetrazoles, tetrazoles, benzofurans, dibenzofurans, benzothiophenes, dibenzothiophenes, thiadiazoles, pyridines, pyrimidines, pyrazines, pyridazines, piperazines, piperidines, morpholines, pyranes, anolines, quinolines, phthalates and combinations of these. The basic structure of the cation can be substituted or not substituted in a single or multiple way.

The anionic portion of the ionic liquid may comprise an inorganic portion, an organic portion, or combinations thereof. In preferred embodiments, the anionic moiety comprises one or more selected moieties of halogens, phosphates, alkylphosphates, alkenylphosphates, bis (trifluoromethylsulfonyl) imide (NTf2), BF4-, PF6 ', AsF6 ", NO3", N (CN) 2-, N (SO3CF3) 2 ~ r amino acids, substituted or unsubstituted carborans, perchlorates, pseudohalogens such as thiocyanate and cyanate, Lewis acids based on metal chloride (for example, zinc chlorides and aluminum chlorides) or C1-6 carboxylates. Pseudohalides are monovalent and have properties similar to those of halides. Examples of pseudohalides useful according to the invention include cyanides, thiocyanates, cyanates, fulminates and azides. Exemplary carboxylates containing 1-6 carbon atoms are formate, acetate, propionate, butyrate, hexanoate, maleate, fumarate, oxalate, lactate, pyruvate, and the like.

Various ionic liquids can be prepared and used in accordance with the present invention. In particular, any combination of the cations and anions mentioned above could be used. It is only necessary to combine one or more cations (for example, those described above) with one or more anions (for example, those described above) to form a material that is liquid under the conditions described herein. For example, an imidazolium cation portion could be combined with an anionic halogen portion to form a material that is liquid under the necessary conditions (for example, 1-butyl-3-methylimidazole chloride) and which is formed substantially completely by ionic portions. Thus, it is clear that the present invention encompasses the use of a wide variety of ionic liquids. Specific non-limiting examples of ionic liquids for use according to the invention include l-butyl-3-methyl-imidazolium chloride ("BmimCl"); 1-allyl-3-methyl-imidazolium chloride ("AmimCl"); 1-ethyl-3-methyl-imidazolium chloride; l-hydrogen-3-methylimidazolium chloride; 1-benzyl-3-methyl-imidazolium chloride ("BenzylmimCl"); 1-isopropyl-3-methyl-imidazolium chloride; 1-m-methoxybenzyl-3-methyl-imidazolium chloride ("MethoxyBenzylmimCl"); 1-m-methylbenzyl-3-methylimidazolium chloride ("MethylBenzylmimCl"); l-benzyl-3-methyl-imidazolium chloride and l-methyl-3-benzyl-imidazolium dicyanide ("Benzylimim").

The invention also encompasses the use of various mixtures of ionic liquids. In fact, mixtures of ionic liquids can be useful for the supply of ionic liquids that have customized properties, for example, viscosity. For example, BenzylmimCl is a relatively viscous ionic liquid; however, its viscosity can be significantly reduced by mixing with AmimCl. The viscosity of the mixture of ionic liquids can therefore be adjusted by varying the proportion between the most viscous component and the least viscous component.

Ionic liquids for use according to the invention can be synthesized according to the literature. Preferably, the ionic liquids are dried (for example, at 100 ° C) in a vacuum oven over a period of time, for example, about 48 hours, before use. In one embodiment, the ionic liquid is formed by a material that is solid (for example, crystalline) under ambient conditions, but is liquid at an increased temperature (for example, greater than about 30 ° C, greater than about 50 ° C, greater than about 75 ° C, or greater than about 100 ° C). Generally, the crystalline material can be placed in an appropriate container and heated until dissolved (see, for example, ionic liquids in "Synthesis", Wasserscheid, P. and Weldon, T. (Eds.), Wiley Pub.). Of course, the ionic liquid can also comprise a material that is liquid under ambient conditions (for example, at a temperature around 20-25 ° C).

Filtration and / or chromatography Solubilized silk proteins can be concentrated and separated from impurities, for example, from the surfactant, ionic liquid and other cellular components, based on charge, hydrophilicity, affinity, solubility or stability, or size. Non-limiting examples of separation techniques include ammonium sulfate precipitation, chromatography and membrane filtration (including membrane filtration by tangential flow). In modalities that use chromatography for separation, exemplary methods include ion exchange (cationic or anionic) chromatography, affinity, hydrophilic interaction, hydrophobic interaction, size exclusion and gel permeation (see US 6,248,570).

In some embodiments, the separation can be carried out by membrane filtration, which includes, without limitation, methods such as, for example, single-pass filtration, dead-end filtration, direct flow filtration (DFF) and cross-flow or flow filtration tangential (TFF). According to the invention, filtration is based on the principle of separating molecules according to size using a semipermeable membrane of a defined range of pore sizes. It is known to those skilled in the art that combinations of filtration methods and types of membrane can be used in the separation.

According to the invention, membrane filtration is the separation of cellular components carried out by polymeric or inorganic membranes. Within the technique, there are four commonly accepted categories of membranes defined by the size of the material they remove from the transport liquid. Methods for sequentially filtering through membranes from the smallest to the largest pore size are reverse osmosis (RO), nanofiltration (NF), ultrafiltration (UF) and microfiltration (MF).

Filtration with the membranes mentioned above separates molecules according to their molecular weight using membranes with specific pore sizes. For example, separation with RO membranes that have pore sizes less than 0.001 micrometer is intended to separate molecules that have a molecular weight less than 200 Daltons. Filtration with NF membranes that have pore sizes of 0.001-0.008 micrometer, inclusive, is intended to separate molecules that have a molecular weight of 200 Daltons to 15 kilodaltons (kDa), inclusive. Filtration with UF membranes that have pore sizes of 0.005-0.1 micrometer, is intended to separate molecules that have a molecular weight of 5 kDa-300 kDa, inclusive. Filtration with microfiltration membranes that have pore sizes of 0.05-3.0 micrometers, inclusive, is intended to separate molecules that have a molecular weight of 100 kDa-3,000 kDa and larger. According to this invention, membrane filtration can separate solubilized silk proteins from other components based on size exclusion using membranes that have a particular molecular weight cut-off value (MQWCO) that is determined by the pore size of the membrane. MWCO, also known as the nominal molecular weight limit (NMWL) or the nominal molecular weight cut-off value (NMWCO), is the size designation in kilodaltons for membrane filtration. MWCO is defined as the molecular weight of the molecule that is 90% retained by the membrane. As, for example, molecules of the same molecular weight can have significantly different shapes, MWCO is not an exact metric, but it is nevertheless a useful metric and is commonly used by filter manufacturers. Both hydrophobic membranes and hydrophilic membranes can be used in the present invention. These membranes can be used as flat blades or in a spiral configuration. Hollow fibers can also be used. Regarding UF membrane compositions, any of several potential membrane materials can be used, including, without limitation, regenerated cellulose, polyether sulfone (which may or may not be modified to alter its inherent hydrophilia), polyvinylidene fluoride and aggregates ceramic and metal oxide. Many UF polyether sulfone membranes can tolerate a pH range of 0.5-13, and temperatures ranging up to 85 ° C. Materials for MF membranes include all used for UF membranes, in addition to polycarbonate, polypropylene, polyethylene and PTFE (TEFLON ™). In some embodiments, the solubilized silk proteins can be filtered to separate large cell debris from smaller cell components to prevent cell debris from interfering with the separation and purification steps that involve the use of membranes or chromatography. In these modalities, the permeate comprises the silk proteins and is recovered.

In some embodiments, a membrane that has a suitable MWCO can be used in a separation step. For example, typical silken proteins used in the methods of the invention have an MW of about 30 kDa. In these embodiments, the retentate comprises silk protein and can be recovered.

In a preferred embodiment, tangential flow filtration acts both to diafiltrate and to concentrate silk proteins. In TFF, the solution typically flows parallel to the filter membrane. A differential pressure across the membrane causes filterable fluid and solutes (whose molecular weight is less than that of the membranes or behaves in this way, for example, globular proteins) to flow through the filter. In HPTFF (high performance tangential flow filtration), the membrane is loaded, thus using both the size and the charge of the molecules to separate contaminants (see US 20030229212). According to the invention, diafiltration can be discontinuous or continuous diafiltration. In batch diafiltration, the solution is concentrated, and the lost volume is replaced with a new buffer. In continuous diafiltration, the volume of the solution is maintained by the inflow of new buffer solution, while the old buffer solution is removed. In some embodiments, the separation and purification of silk proteins can be accomplished by tangential flow filtration methods using ultrafiltration membranes.

Uses The silk dope produced using the methods of the invention can be used for a wide and diverse set of medical, military, industrial and commercial applications. For example, silk dope is used to produce silk fibers, which, in turn, can be used in the manufacture of medical devices such as sutures, skin grafts, cell growth matrices, ligament replacement and surgical mesh, and in a wide range of industrial and commercial products such as cables, ropes, nets, fishing lines, clothing fabrics, bulletproof vest lining, container fabrics, backpacks, handbag handles or wallets, adhesive binder material, non-adhesive binder material, mooring material, tent fabric, oilcloths, pool covers, vehicle covers, fence material, sealants, construction material, waterproof material, flexible partition material , sports equipment; and, in fact, in almost all uses of fibers or fabrics for which high tensile strength and elasticity are desired characteristics. The silk dope can also have applications for use in the production of compositions for personal hygiene products, such as cosmetics, skin care, hair care and hair coloring; and in the coating of particles, for example, pigments.

Silks can be used in their native form or can be modified to form derivatives, which provide a more beneficial effect. For example, silks can be modified by conjugation to a polymer to reduce allergenic properties, as described in US 5,981,718 and US 5,856,451. Suitable modifying polymers include, without limitation, polyalkylene oxides, polyvinyl alcohol, polycarboxylates, poly (vinylpyrrolidone) and dextrans. In another example, silks can be modified by selective digestion and joining other protein modifiers. For example, silk proteins can be cleaved into smaller peptide units by treatment with acid at an elevated temperature of about 60 ° C. Useful acids include, without limitation, dilute hydrochloric acid, sulfuric acid or phosphoric acid. Alternatively, the digestion of silk proteins can be done by treatment with a base, for example, sodium hydroxide, or enzymatic digestion can be done using a suitable protease.

Proteins can also be modified to provide performance characteristics that are beneficial in specific applications for personal care products. The modification of proteins for use in personal care products is well known in the art. For example, commonly used methods are described in US 6,303,752, US 6,284,246. and US 6,358,501. Examples of modifications include, without limitation, ethoxylation to promote increased water-oil emulsion, siloxylation to provide lipophilic compatibility, and esterification to aid compatibility with soap and detergent compositions.

In addition, silk proteins can be derivatized with functional groups that include, without limitation, amines, oxirans, cyanates, carboxylic acid esters, silicone copolyols, siloxane esters, quaternized amine aliphatics, urethanes, polyacrylamides, dicarboxylic acid esters and halogenated esters. Silk proteins can also be derivatized by reaction with diimines and by the formation of metal salts.

Consistent with the definitions of "polypeptide" (and "protein") above, these derivatized and / or modified molecules are also broadly referred to herein as "polypeptides" and "proteins". The silk dope can be spun together and / or bundled or braided with other types of fiber. Examples include, without limitation, polymeric fibers (for example, polypropylene, nylon, polyester), fibers and silks from other plants and animal sources (for example, cotton, wool, Bombyx mori or spider silk) and glass fibers. A preferred embodiment is silk fiber braided with 10% polypropylene fiber. The present invention contemplates that the production of such combinations of fibers can be easily practiced to enhance any desired characteristics, for example, appearance, softness, weight, durability, water repellency properties, advantageous manufacturing cost, which can generally be sought in manufacturing and production of fibers for medical, industrial or commercial applications.

Personal care products Cosmetic and skin care compositions can be anhydrous compositions that comprise an effective amount of silk in a cosmetically acceptable medium. The uses of these compositions include, without limitation, skin care, skin cleansing, makeup and anti-wrinkle products. An effective amount of silk for cosmetic and skin care compositions is defined herein as a ratio of about 10-4 to about 30% by weight, but preferably about 10-3 to 15% by weight relative to the total weight of the composition. This proportion may vary depending on the type of cosmetic composition or for skin care. Compositions suitable for a cosmetically acceptable medium are described in US 6,280,747. For example, the cosmetically acceptable medium may contain a grease substance in a proportion generally from about 10 to about 90% by weight relative to the total weight of the composition, where the grease phase contains at least one liquid, solid or grease substance. semi-solid. The grease substance includes, without limitation, oils, waxes, gums and so-called pasty grease substances. Alternatively, the compositions can be in the form of a stable dispersion such as, for example, a water-in-oil or oil-in-water emulsion. In addition, the compositions may contain one or more conventional cosmetic or dermatological additives or adjuvants, including, without limitation, antioxidants, preservatives, fillers, surfactants, UVA and / or UVB sunscreens, fragrances, thickeners, wetting agents and anionic polymers, not ionic or amphoteric, and dyes or pigments.

Emulsified cosmetics and almost drugs that can be produced using emulsified materials that comprise silk produced by a method of the invention, for example, cleaning cosmetics (beauty soap, facial wash, shampoo, creme-rinse, and the like), products for hair care (fake hair, hair cosmetics, and the like), basic cosmetics (general cream, emulsion, shaving cream, conditioner, colognes, shaving lotion, cosmetic oil, face mask, and the like), cosmetics makeup (foundation, eyebrow pencil, eye cream, eye shadow, mascara, and the like), aromatic cosmetics (perfume and the like), tanning and sun protection cosmetics (tanning and sun protection cream, tanning lotion and sun protection, tanning oil and sun protection, and the like), nail cosmetics (nail cream, and the like), eyeliner cosmetics (eyeliner, and the like), cosmetics lip cosmetics (lipsticks, lip cream, and the like), oral care products (toothpaste, and the like), bath cosmetics (bath products, and the like), and the like.

The cosmetic composition can also be in the form of nail care products, for example, nail polish. Nail polishes are defined here as compositions for the treatment and coloring of nails, which comprise an effective amount of silk in a cosmetically acceptable medium. An effective amount of silk for use in a nail polish composition is hereby defined as a ratio of about 10 "4 to about 30% by weight to the total weight of the nail polish.

Components of a cosmetically acceptable medium for nail polish are described in US 6,280,747. Nail polish typically contains a solvent and a film-forming substance, for example cellulose derivatives, polyvinyl derivatives, acrylic polymers or copolymers, vinyl copolymers and polyester polymers. The composition can also contain an organic or inorganic pigment.

Compositions for hair care are defined herein as compositions for the treatment of hair, including, without limitation, shampoos, conditioners, lotions, aerosols, gels and mousses, which comprise an effective amount of silk in a cosmetically acceptable medium. An effective amount of silk for use in a hair care composition is hereby defined as a ratio of about 10 "2 to about 90% by weight to the total weight of the composition. Components of a cosmetically acceptable medium for hair care compositions are described in US 2004/0170590, US 6,280,747, US 6,139,851 and US 6,013,250. For example, such hair care compositions can be aqueous, alcoholic or aqueous-alcoholic solutions, the alcohol preferably being ethanol or isopropanol, in a proportion of about 1 to about 75% by weight relative to the total weight, for aqueous-alcoholic solutions. In addition, hair care compositions may contain one or more conventional cosmetic or dermatological additives or adjuvants, as shown above.

Compositions for coloring hair are defined herein as compositions for coloring, shaming or lightening hair, which comprise an effective amount of silk in a cosmetically acceptable medium. An effective amount of silk for use in a hair coloring composition is hereby defined as a ratio of about 10'4 to about 60% by weight to the total weight of the composition. Components of a cosmetically acceptable medium for hair coloring compositions are described in US 2004/0170590, US 6,398,821 and US 6,129,770. For example, hair coloring compositions often contain a mixture of peroxygen-based inorganic oxidizing dye and an oxidizable dyeing agent. The oxidizing coloring agent based on peroxygen is most commonly hydrogen peroxide. Oxidizing agents for hair coloring are formed by oxidative coupling of primary intermediates (for example, p-phenylenediamines, p-aminophenols, p-diaminopyridines, hydroxyindols, aminoindols, aminothymidines or cyanophenols) with secondary intermediates (for example, phenols, resorcinols , m-aminophenols, m-phenylenediamines, naphthols, pyrazolones, hydroxyindoles, catechols or pyrazoles). Additionally, hair coloring compositions may contain oxidizing, sequestering, stabilizing, thickener, carrier-buffer, surfactants, solvents, antioxidants, polymers, non-oxidative dyes and conditioners.

Silks can also be used to coat pigments and cosmetic particles in order to increase the dispersibility of the particles for use in cosmetic and coating compositions. Cosmetic particles are defined herein as particulate materials such as, for example, pigments or inert particles, which are used in cosmetic compositions. Suitable cosmetic pigments and particles include, without limitation, inorganic colored pigments, organic pigments and inert particles. Inorganic colored pigments include, without limitation, titanium dioxide, zinc oxide and oxides of iron, magnesium, cobalt; and aluminum. Organic pigments include, without limitation, Red D&C N ° 36, Orange D&C N ° 17, the calcium lakes of Red D&C Nos 7, 11, 31 and 34, the barium lake of Red D&C N ° 12, the lake of strontium of Red D&C N ° 13, the aluminum lake of Yellow FD&C N ° 5 and black carbon particles. Inert particles include, without limitation, calcium carbonate, aluminum silicate, calcium silicate, magnesium silicate, mica, talc, barium sulfate, calcium sulfate, powdered nylon, perfluorinated alkanes and other inert plastics.

Silks can also be used in dental floss (see, for example, US 2005/0161058). The dental floss may be monofilament yarn or multifilament yarn, and the fibers may or may not be twisted. The dental floss can be packed as individual pieces or in a roll with a cutter to cut pieces to any desired length. Dental floss can be supplied in several formats in addition to filaments, for example, without limitation, tapes and blades, and the like. The dental floss can be coated with different materials, for example, without limitation, wax, polytetrafluoroethylene monofilament yarn for dental floss. Silks can also be used in soaps (see, for example, US 2005/0130857).

Pigment and cosmetic particle coating The effective amount of silk for use in pigment and cosmetic particle coating is defined here as a ratio of about 10 "4 to about 50%, but preferably about 0.25 to about 15% by weight in relation to the dry particle weight. The optimum amount of silk to be used depends on the type of pigment or cosmetic particle being coated. For example, the amount of silk used with inorganic colored pigments is preferably between about 0.01% and 20% by weight. In the case of organic pigments, the preferred amount of silk is between about 1% to about 15% by weight, while, for inert particles, the preferred amount is between about 0.25% to about 3% by weight. Methods for preparing coated pigments and particles are described in US 5,643,672. These methods include: adding an aqueous solution of the silk to the particles during stirring or mixing, forming a silk and particle broth and drying, spray drying a solution of the silk on the particles or freeze-drying a silk and particle broth. particles. These coated pigments and cosmetic particles can be used in cosmetic formulations, dyes, inks, and the like.

Biomedical Silks can be used as a dressing on a bandage to promote wound healing. For this application, the bandage material is coated with an effective amount of silk. For a wound healing bandage, an effective amount of silk is defined herein as a proportion of about 10-4 to about 30% by weight relative to the weight of the bandage material. The material to be coated can be any biologically inert soft porous fiber or fabric. Examples include, without limitation, cotton, silk, rayon, acetate, acrylic, polyethylene, polyester, and combinations thereof. The coating of the fabric or fiber can be obtained by several methods known in the art. For example, the material to be coated can be immersed in an aqueous solution containing the silk. Alternatively, the solution containing the silk can be sprayed onto the surface of the material to be coated using a spray gun. In addition, the solution containing the silk can be coated on the surface using a cylinder coating printing process. Wound bandaging may include other additives, including, without limitation, disinfectants such as, for example, iodine, potassium iodide, povidone iodine, acrinol, hydrogen peroxide, benzalkonium chloride and chlorhexidine; healing accelerating agents such as, for example, allantoin, dibucaine hydrochloride and chlorophenylamine malate; vasoconstrictive agents, such as naphazoline hydrochloride; astringent agents, for example, zinc oxide; and crusting agents such as, for example, boric acid.

The silk dope can also be used in the form of a film as a wound dressing material. The use of silk, in the form of an amorphous film, as a wound dressing material, is described in US 6,175,053. The amorphous film comprises a dense, non-porous film with a crystallinity below 10% which contains an effective amount of silk. For a wound care film, an effective amount of silk is defined here as between about 1 and 99% by weight. The film can also contain other components, including, without limitation, other proteins such as, for example, sericin, and disinfectants, healing accelerating agents, vasoconstricting agents, astringent agents and scabing agents, as described above. Other proteins, such as sericin, can comprise 1 to 99% by weight of the composition. The amount of the other ingredients listed is preferably below a total of about 30% by weight, more preferably between about 0.5 and 20% by weight of the composition. The wound dressing film can be prepared by dissolving the aforementioned materials in an aqueous solution, removing insolubles by filtration or centrifugation, and molding the solution on a solid smooth surface, such as an acrylic plate, followed by drying. .

The silk dope can also be used to produce sutures (see, for example, US 2005/0055051). These sutures may feature a braided jacket made of fibers and ultra-high molecular weight silk fibers. Polyethylene provides strength. Polyester fibers can be braided with the weighted molecular weight polyethylene to provide enhanced binding properties. Silk can be supplied in a contrasting color to provide a signal to increase suture recognition and identification. Silk is also more compliant to the fabric than other fibers, allowing the ends to be cut closer to the knot, without concerns of deleterious interaction between the ends of the suture and the surrounding tissue. The handling properties of the high-strength suture can also be increased with the use of various materials to coat the suture. The suture advantageously has the strength of the No. 5 Ethibond suture, although it still has the diameter, feel and ability to tie the No. 2 suture. As a result, the suture is ideal for most orthopedic procedures such as, for example, repair of the suture. rotator cuff, Achilles tendon repair, patellar tendon repair, ACL / PCL reconstruction, hip and shoulder reconstruction procedures, and replacement for sutures used in or with suture anchors. The suture may not be coated, or be coated with wax (beeswax, petroleum wax, polyethylene wax, or others), silicone (Dow Corning 202A silicone fluid, OR others), silicone rubbers, PBA (polybutylate acid) ), ethyl cellulose (Filodel) or other coatings, to increase the lubricating properties of the yarn, safety of the knot or resistance to abrasion, for example.

The silk dope can also be used to produce stents (see, for example, US 2004/0199241). For example, an endoprosthesis graft is provided that includes an endoluminal endoprosthesis and a graft, wherein the endoprosthesis graft includes silk. Silk induces a response in a host receiving the endoprosthesis graft, in which the response can lead to increased adhesion between the silk endoprosthesis graft and the host tissue that is adjacent to the silk of the silk endoprosthesis graft. Silk can be attached to the graft in various ways, for example, by interweaving the silk into the graft or by adhering the silk to the graft (for example, by means of an adhesive or by suturing). The silk can be in the form of a thread, a braid, a blade, dust, etc. As for the location of the silk in the endoprosthesis graft, the silk can be attached only to the outside of the endoprosthesis, and / or the silk can be attached to the distal regions of the endoprosthesis graft, in order to help fix these distal regions to the neighboring tissue in the host. A wide variety of stent grafts can be used within the context of the present invention, depending on the location and nature of the desired treatment. Endoprosthesis grafts can be, for example, bifurcated or tube grafts, cylindrical or narrowed, self-expanding or balloon-expandable, with a single or modular body, etc.

In addition to silk, the endoprosthesis graft may contain a coating on part or all of the silk, whereby the coating degrades after insertion of the endoprosthesis graft into a host, with the coating thereby delaying contact between the silk and the host. Adjacent coatings include, without limitation, gelatin, degradable polyesters (for example, PLGA, PLA, MePEG-PLGA, PLGA-PEG-PLGA, and copolymers and mixtures thereof), cellulose and cellulose derivatives (for example, hydroxypropyl cellulose), polysaccharides (for example, hyaluronic acid, dextran, dextran sulfate, chitosan), lipids, fatty acids, sugar esters, nucleic acid esters, polyanhydrides, polyorthoesters and polyvinyl alcohol (PVA). Endoprosthesis grafts that contain silk may contain a biologically active agent (drug), in which the agent is released by the endoprosthesis graft and then induces an increased cellular response (for example, deposition of cellular or extracellular matrix) and / or fibrotic response in a host in which the stent graft was inserted. The silk dope can also be used to produce a matrix for the production of ligaments and tendons ex vivo (see, for example, US 2005/0089552). A silk fiber-based matrix can be seeded with pluripotent cells, for example, bone marrow stromal cells (BMSCs). The ligament or tendon created by bioengineering is advantageously characterized by a cellular orientation and / or wavy pattern of the matrix in the direction of the applied mechanical forces, and also by the production of specific markers for ligament and tendon, including type I collagen, type III collagen and proteins of fibronectin along the axis of the mechanical load produced by mechanical forces or stimulation, if these forces were applied. In a preferred embodiment, the ligament or tendon is characterized by the presence of bundles of fibers that are arranged in a helical organization. Some examples of ligaments or tendons that can be produced include anterior cruciform ligament, posterior cruciform ligament, rotator cuff tendons, medial collateral ligament of the elbow and knee, flexor tendons of the hand, lateral ligaments of the ankles, and tendons and ligaments of the jaw or joint temporomandibular joint. Other tissues that can be produced by methods of the present invention include cartilage (both articular and meniscal), bone, muscle, skin and blood vessels. The silk dope can also be used to produce hydrogels (see, for example, US 2005/0266992). Silk fibroin hydrogels can be characterized by an open pore structure that allows its use as 5 frameworks for tissue engineering, substrate for cell culture, dressing for wounds and burns, substitutes for soft tissues, bone filling, in addition to support for pharmaceutical or biologically active compounds. 10 The silk dope can also be used to produce dermatological compositions (see, for example, US 2005/0019297). In addition, dope can also be used to produce sustained release compositions (see, for example, US 2004/0005363). 15 Textiles

The silk dope can also be used to produce a coating for the fiber surface for subsequent use in textiles. This provides a monolayer of the protein film on the fiber, resulting in a smooth finish. US 6,416,558 and US 5,232,611 describe the addition of a finish coat to the fibers. The methods described in these disclosures provide examples of the versatility of the fiber finish to provide a good touch and a smooth surface. For this application, the fiber is coated with an effective amount of silk. For the purpose of fiber coating for use in textiles, an effective amount of silk is defined herein as a ratio of about 1 to about 99% by weight to the weight of the fiber material. Fiber materials include, without limitation, cotton textile fibers, polyesters such as rayon and Lycra ™, nylon, wool, and other natural fibers, including native silk. Compositions suitable for the application of silk on the fiber can include co-solvents such as, for example, ethanol, isopropanol, hexafluoranols, isothiocyanouranates, and other polar solvents that can be mixed with water to form solutions or microemulsions. The solution containing silk can be sprayed onto the fiber or the fiber can be immersed in the solution. Although not necessary, instant drying of the coated material is preferred. An alternative protocol is the application of the silk composition on braided fibers. An ideal modality of this request is the use of silks to cover stretch fabrics, such as those used for pantyhose.

Composite materials Silk fibers can be added to polyurethane, other resins or thermoplastic fillers to prepare panels for panels and other construction materials, or as furniture and moldable worktops that replace wood and particle boards. The compounds can also be used in the construction and automotive industry, especially ridges and door panels. Silk fibers reinforce the resin making the material much stronger and allowing for a lighter weight construction that has equal or greater resistance to other particle boards and composite materials. Silk fibers can be isolated and added to a synthetic compound-forming resin, or used in combination with plant-derived proteins, starch and oils to produce biologically based composite materials. Processes for producing these materials are described in JP 2004284246, US 2005175825, US 4,515,737, JP 47020312 and WO 2005/017004. Additives for paper The properties of silk fiber can add strength and quality texture to paper production. Tissue papers are made by mixing silk threads in cotton pulp to prepare very smooth handmade papers used for wrapping gifts, notebook covers, bags. Processes for producing paper products from silk dope are generally described in JP 2000139755.

Advanced materials Silks produced from the silk dope of the invention have considerable hardness and stand out among other silks in maintaining these properties when wet (Hepburn et al, 1979).

Areas' of substantial growth in the clothing textile industry are technical and intelligent textiles. There is a growing demand for healthy textile products, with high functional value, environmentally friendly and personalized. Fibers, such as those of the invention, which do not alter properties when joined and, in particular, maintain their strength and extensibility, are useful for functional sports and leisure clothing, as well as work clothing and protective clothing.

Developments in weapon and surveillance technologies are creating innovations in personal protective equipment and battlefield-related systems and structures. In addition to conventional requirements such as, for example, durability of the material from prolonged exposure, insulation and protection from the external environment, silk textiles produced from the silk dope of the invention can be processed to resist ballistic projectiles, fire and chemical substances. Processes for producing these materials are described in WO 2005/045122 and US 2005268443.

EXAMPLES EXAMPLE 1 - Recombinant production and purification of bee silk proteins To create recombinant expression constructs, the four sequences of the bee silk gene (Genbank Access Nos: FJ235088; FJ235089, FJ235090, FJ235091) without signal peptides were amplified by PCR from the cDNA clones described in Sutherland et al. (2006) using the following sets of oligonucleotide primers:

AmelFl: GGAATT CTC ATG A.GT TTG GAG GGG CCG GGC AAC TCG (SEQ ID NO: 101) and CGGC GGATCC TTA TTA AAA TAC GTT GCT CTT CAA GT (SEQ ID NO: 102);

AmelF2: GGAATT CTC ATG AGC CGC GTG ATT AAT CAC GAG TCG CTG (SEQ ID NO: 103) and CGGC GGATCC TTA TTA TTC CAA CTT TGC TAC ATG TAT TTT C (SEQ ID: 104) ;

AmelF3: GGAATT CCC ATG GGC GTC GAG GAA TTC AAG TCC TCG (SEQ ID NO: 105) and CGGC AGATCT TTA TTA AAA TTT TIT ATC CTC AAT A (SEQ ID NO: 106);

AmelF4: GGAATT CCC ATG GCA AGG GAA GAG GTG GAG ACA CGG (SEQ ID NO: 107) and CGGC GGATCC TTA TTA CTT CAC CTC CCA TTC TTC ATT C (SEQ ID NO: 108) (sites of restriction enzyme cloning are underlined and in bold and sequences that match the cDNA sequence are shown in italics).

The PCR amplicons were cloned into restriction enzyme sites (AmelFl and AmelF2: BspHl and Bam HI; AmelF3: Ncol and Bgl II; AmelF4: Ncol and Bam HI) of the expression vector pET14b (Novagen) and the sequences checked for sequencing of DNA before expression.

The constructs were transformed into competent Rosetta 2 (DE3) cells (Novagen) and silk proteins were initially expressed in 50 ml of TB medium of instant expression overnight (Novagen) in shaking flasks. The four bee silk proteins, AmelFl-4, were synthesized in E. coli cells in soluble form at 20 ° C and in insoluble form at 30 ° C and 37 ° C. The highest protein yields, assessed by comparative protein band intensity after SDS-PAGE (Figure 1), were obtained when expression was performed for prolonged periods (24-36 h) at temperatures> 30 ° C with the proteins recovered from inclusion bodies. Quantitative analysis of the gel band intensity, with protein identities confirmed by mass spectroscopy, indicated that the protein recovered from the inclusion bodies was basically pure silk protein (> 95%). The subsequent analysis found that the solubilized proteins of the inclusion bodies self-assembled in a native type structure. In this way, all subsequent expression was performed under conditions such that the recombinant proteins were recovered from the inclusion bodies.

In order to increase protein yield, a batch-fed batch fermentation process was developed and optimized for AmelF3. Fermentations were carried out in 2-liter Biostat B culture vessels (Sartorius Stedim, Melsungen, Germany) using minimal medium (1.6 liter starting volume). Glucose was used as an initial carbon source, switching to a glycerol feed after inducing the expression of silk protein with IPTG. The starting medium contained (per liter): KH2PO4, 13.3 g; (NH4) 2HPO4, 4 g and citric acid 1.7 g. The pH of the medium was adjusted to a final value of 7.0 using 2 M NaOH. The following components were sterilized separately, and then added (per liter of final medium): 40 ml of 50% glucose (w / v); 5 ml of 1 M MgSO4; 130 μl of 0.1 M thiamine hydrochloride; 1 ml of 100 mg / ml ampicillin and 5 ml of a vitamin / trace metal solution containing (per liter of solution): biotin, 0.2 g; CuSO4.5 H2O, 2.0 g; Nal, 0.08 g; MnSO4.H2O, 3.0 g; Na2MoQ4.2 H2O, 0.2 g; boric acid, 0.02 g; CoCl2.6 H2O, 0.5 g; ZnCl2, 7.0 g; FeSO4.7 H2O, 22.0 g; CaSO4.2 H2O, 0.5 g and H2SO4, 1 ml.

The fermentation inocula for the four strains were grown for 20 h at 37 ° C in the same medium used in the fermenter. After the inocula were transferred to the fermentation vessels, the pH of the medium was adjusted and controlled to 7.0 by adding NH4OH 10% (w / v) or H3PO4 10% (w / v). The temperature was controlled at 37 ° C, and the concentration of dissolved oxygen (DO) was maintained above 40% of air saturation by manipulating the stirring speed up to 1,100 rpm and enriching the air supply with pure oxygen, when necessary.

When the cultures had grown to an optical density value at 600 nm (OD60onm) of approximately 20 (approximately 10 h after inoculation), IPTG was added to a final concentration of 1 mM. The fermenters were operated in batch mode until all glucose was consumed, as indicated by a sharp rise in OD (-12 h after inoculation). In the batch phase fed with glycerol, 400 ml of 62% glycerol solution (v / v) were fed into the fermenter at a rate of 50 ml / h. The cultures were developed for 24 h, when the cells were then collected by centrifugation and stored at - 80 ° C. Under these conditions, the yeast OD6oonm value was 34 and the yield of purified recombinant AmelF3 after solubilization was approximately 2.5 grams per liter of yeast. The same fermentation conditions were used to express the other bee silk proteins. The strains expressing AmelFl, 2 and 4 silk proteins grew to OD6oonm values of 30, 67 and 57, respectively. The yields of purified recombinant proteins AmelFl, 2 and 4 after solubilization were approximately 0.2, 1.5 and 1.9 grams per liter of yeast, respectively.

The yield of 2.5 g / 1 of purified protein from the optimized fed batch fermentation system is by far the highest level of expression reported for any recombinant silk protein. Factors that contribute to this high yield include the size and nature of the bee fibroin genes and the structural properties of silk proteins. In contrast to the large size (> 10 kbp) and the highly repetitive nature of the genes encoding well-studied excavated spider silk and silkworm cocoon silk, bee silk genes are small (approximately 1 kbp ) with much less repetition in their DNA sequences (Sutherland et al, 2006). The smaller size and reduced level of repetition mean that bee genes are not prone to genetic instabilities that include premature termination of translation and truncation that result from the transgenic expression of highly repetitive nucleotide sequences.

The silk proteins in inclusion bodies were purified from E. coli cells after repeated treatment with BugBuster Master Mix (Novagen), according to the manufacturer's protocol for preparing soluble or inclusion bodies. Protein solutions were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) with a 4-12% gradient (Invitrogen). The identification of recombinant silk protein was verified by tandem mass spectrometry, as previously described (Sutherland et al, 2006).

The silk proteins in inclusion bodies were solubilized in 3% sodium dodecyl sulfate (SDS) with incubation for 2 h at 60 ° C. The protein concentration in solution was measured using a BCA QuantiPro assay kit (Sigma). When necessary, solutions of each of the four recombinant bee silk proteins were mixed in equimolar proportions. Excess SDS was removed from protein solutions by dialysis against 5 g / 1 KC1 solution causing KDS precipitation. The precipitate was removed by centrifugation at 1.6000 g for 5 min. EXAMPLE 2 - Fourier transform infrared spectroscopy (FTIR) Infrared spectroscopy with Fourier transform

Fourier was used to compare the protein structure of native and recombinant bee silks. Blades of native bee silk were obtained from a commercial hive, washed intensely in chloroform to remove wax and washed intensely in warm water to remove water-soluble contaminants. Solutions of each of the four recombinant bee silk proteins were mixed in equimolar proportions, molded and dried on a film. Infrared spectra of these samples were obtained in the transmission mode using a Perkin-Elmer System 2000 Fourier transform spectrometer adapted with a series i infrared image microscope accessory. The spectra were collected using the Spectrum software (version 5.3.1) and represent the average of 256 scans collected at a resolution of 4 cm "1. The manipulation and analysis of post-collection data were performed using the Grams / AI v5 software .05 The deconvolution of the amide I region for each silk spectrum is shown in Figure 2. A summary of the results and the secondary structure designations of the component are shown in Table 2. Table 2. Summary of the FTIR fit curve.

The FTIR results suggest that native bee silk contains approximately 65% of coiled-coil structure, which is consistent with previous predictions based on sequence (Sutherland et al, 2006). The spectrum of recombinant silk is very similar to the spectrum of native silk, and it is estimated that the recombinant silk contains 59% coiled-coil structure. EXAMPLE 3 - Dry wiring

Inclusion bodies were solubilized in 3% SDS to generate soluble bee fibroin solutions generally between 0.5-2% by weight of protein and up to 3% by weight of protein. The excess SDS was removed from the silk protein solutions by precipitation of KC1. The potassium precipitation removed up to 95%, for example, 70-80%, of SDS (weighing the precipitate), but <10% of the protein (by measuring the protein concentrations in solution). The silk solutions were concentrated by prolonged dialysis against 20% of the weight of polyethylene glycol (PEG, MW 8000, Sigma) or Slide-A-Lyzer concentration solution (Pierce), until a honey-like viscosity was obtained (in 10-15% of the protein weight). A droplet of concentrated silk dope was suspended between the teeth of a pair of tweezers in air and the tweezers were opened to form a fine thread (Figure 3A). These single-stranded wires were stable in the air, but dissolved in water. The fibers were then submerged in a 90% methanol - 10% water bath, extracted a second time up to approximately 2x the length, and air dried (Figure 3B). The double-stranded threads were not water-soluble. The single-stranded and double-stranded fibers were examined by an optical microscope with polarizing lenses, and by a Zeiss EVO LS environmental scanning electron microscope.

The recombinant silk threads with ESEM images (not shown) were circular in cross-section and sufficiently uniform in diameter along their length. The single extract fibers had small bodies that adhered to their surface that could be salt crystals; however, the double-stranded fibers had smooth surfaces. Polarized light microscopy showed that single-stranded fibers are not birefringent, but double-stranded fibers are strongly birefringent (Figure 3).

EXEMPLO 4 - Fiação a úmidoSingle-stranded and double-stranded fibers and recombinant silk films were analyzed by wide-angle X-ray scattering in the "Australian Synchrotron" SAXS / WAXS beamline. A wavelength of 0.88 6 A and a camera length of 0.558 m provided a q amplitude of approximately 0.07 to 1.4 Â "\ which was calibrated using a silver behenate standard. WAXS standards for the film and for single-stranded fibers were dominated by a strong signal from SDS crystals, but this was not detectable in double-stranded fibers. The present inventors therefore calculate that double-stranded yarns contain <0.1% of crystals of SDS per unit length found in double-stranded yarns.The dispersion patterns of recombinant silk protein could not be analyzed due to the strong SDS diffraction that limits the sensitivity of the technique, or the low signal-to-noise noise, in the case of very fine double extraction fibers.5 The strength and extensibility of recombinant bee silk threads were measured on an Instron Tensile Tester model 4501 at a rate of 2.5 mm / min. air at 21 ° C and 65% relative humidity. Before testing, each fiber was placed through a 3 mm slit in a plastic frame and fixed with epoxy glue. The measured length (Lo) and the diameter of each fiber were measured using an optical microscope. Table 3 compares the mechanical properties of recombinant silk fibers with the properties of native fibers extracted from the bee silk gland. Table 3. Tensile properties of recombinant bee silk fibers compared to native fibers.

EXAMPLE 4 - Wet spinning

Silk proteins were generally prepared as described in Example 1. Generally, the protein concentration after SDS solubilization was around 3% silk protein. If the protein solutions had a lower concentration, they were concentrated by prolonged dialysis against 20% of the weight of polyethylene glycol (PEG, MW 8000, Sigma) or Slide-A-Lyzer concentration solution (Pierce), until the solutions were 3-6% silk protein.

Concentrated protein solutions of AmelFl-4 or AmelF3 equimolar mixtures alone were extruded through 10 cm of 100 μm capillary tubing at a rate of 10 m / min in methanol solution (50-90% methanol), which caused the formation of a fine, continuous thread. The wires were dried in the air and examined by an optical microscope with polarizing lenses. The strands showed significant birefringence, indicating that the proteins within the strands were directionally aligned (Figure 4A). The air-dried fibers were submerged in a 10% methanol bath of 10% water and extracted a second time to approximately 2x the length (Figure 4B) or 4x the length (Figure 40), and air-dried. The resistance and extensibility of recombinant bee silk threads were measured on an Instron Tensile Tester model 4501 at a rate of 2.5 mm / min. The tests were carried out in the air at 21 ° C and 65% relative humidity. Before the test, each fiber was placed through a 3 mm slit in a plastic frame and fixed with epoxy glue. The measured length (Lo) and the diameter of each fiber were measured using an optical microscope.

Tabela 5. Propriedades mecânicas de fibras de seda de abelha.

EXEMPLO 5 - Dicroismo circular (CD)Tables 4 and 5 describe the mechanical properties of fibers not extracted from recombinant silk. The extraction resulted in threads that were stronger and insoluble in water, and highly birefringent. 5 Table 4. Tensile properties of recombinant bee silk fibers after extrusion of concentrated dope of silk protein in methanol, with and without extraction.

Table 5. Mechanical properties of bee silk fibers.

EXAMPLE 5 - Circular dichroism (CD)

Bee silk protein AmelF3 was expressed in the inclusion bodies of E. coll. AmelF3 inclusion bodies were split using equivalent dry weight of sodium dodecyl sulfate detergent (SDS) to generate 2-4% monomeric protein solutions. The dynamic light scattering (DLS) measured the hydrodynamic diameter of particles in the protein-detergent solution diluted ten times in 100 mM NaCl as 9.2 +/- 0.1 nm (peak containing 98.2% of the particle volume). The diameter of SDS micelles in 3% SDS solutions without protein under the same experimental conditions was a single peak at 5.5 +/- 0.2 nm. No SDS micelles were detected in the SDS-protein solutions, confirming that the majority of the SDS was bound to the protein.

Proteins were refolded by removing SDS using KC1. Potassium dodecyl sulfate had significantly less solubility than SDS and precipitated out of solution where it can be removed by centrifugation. Protein solutions were dialyzed against water to reduce salt levels, and then concentrated by dialysis against PEG8000, resulting in a concentration of 3-4% protein and 0.2-0.4% SDS. When AmelF3 solutions were diluted ten times in 100 mM NaCl (comparable with physiological salt levels), the particle diameter increased to 20.3 +/- 0.7 nm (peak containing 86.8% of the volume of particle), in accordance with the approximate particle diameter calculated for an AmelF3 coiled-coil.

The CD spectra of the bee AmelF3 solutions (0.12%) maintained in a sandwich quartz cell with a 0.01 mm path length (Nova Biotech, El Cajon, CA) were collected using an AVIV Model 410 spectrophotometer ( AVIV Biomedical, Inc., Lakewood, NJ) with a temperature controller. All samples were scanned at 25 ° C, with a bandwidth of 1 nm from 260 nm to 180 nm, and the results were calculated as the average of four repeated experiments. The CD spectra of AmelF3 solutions showed strong spectral minima at 220 and 209 nm and a 220 nm / 209 nm ratio of 1.02, supporting a coiled-coil structure. A 220 nm / 209 nm ratio of one or more is indicative of coiled-coil, while a proportion of less than 0.86 is indicative of isolated propellers. DLS measurements indicated that, after adding SDS back to AmelF3 solutions, the hydrodynamic particle diameter was reduced to the size observed in the original monomeric solutions, confirming that the removal of most SDS is a prerequisite for the protein folds into a conformation of the native type silk protein. In contrast to the AmelF3 protein, His-tagged recombinant versions of the homologous protein of Apis cerana remained monomeric and predominantly with random coil at comparable concentrations (Shi et al., 2008). This result shows that AmelF3 alone, when prepared in the presence of low levels of SDS, folds and adopts a molecular silk structure of the native type.

It will be noted by those skilled in the art that various variations and / or modifications can be made to the invention as shown in the specific modalities, without departing from the spirit or scope of the invention as widely described. The present modalities, therefore, must be considered in all aspects as illustrative, and not restrictive. This application claims priority of US 61 / 237,156 filed on August 26, 2009 and US 61 / 315,812 filed on March 19, 2010, the total contents of which are incorporated herein by reference. All publications discussed and / cited here are incorporated in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification has the sole purpose of providing a context for the present invention. It should not be considered an admission that any or all of these matters form part of the basis of the established technique or were common knowledge with the relevant field for the present invention as if it existed before the priority date of each claim in this application.

REFERENCES Atkins (1967) J. Mol. Biol. 24: 139-41. Delorenzi and Speed (2002) Bioinformatics 18: 617-625. Harayama (1998) Trends Biotech. 16: 76-82. Hepburn et al (1979) Insect Biochem. 9: 69-77. Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453. Sezutzu et al. (2007) Biosci. Biotechnol. Biochem. 71: 2.725-34. Shi et al (2008) Biomaterials 29: 2.820-8. Sutherland et al (2006) Genome Res. 16: 1.414-21. Sutherland et al (2007) Mol. Biol. Evol. 24: 2,424-

Claims (32)

1. Method for producing silk dope comprising silk proteins that have a coiled-coil tertiary structure, the method characterized by comprising: i) lysing cells that produce one or more silk proteins capable of forming a coiled-coil tertiary structure, ii ) solubilize the silk proteins by their contact with an amount of anionic surfactant sufficient to solubilize the silk proteins, and iii) concentrate the silk proteins while reducing the amount of anionic surfactant to produce silk dope, in which the silk dope comprises anionic surfactant and silk proteins that have a coiled-coil tertiary structure, and in which the cells are bacterial cells, yeast cells, insect cells, plant cells or animal cells, or a combination of two or more of these . 2. Method, according to claim 1, characterized in that the silk proteins are concentrated by: a) reducing the amount of surfactant in solution by adding a compound that precipitates the surfactant, and b) separating the solution comprising the proteins from silk from the precipitate formed in step (a) to produce the silk dope. 3. Method, according to claim 1, characterized by the fact that the compound that precipitates the surfactant is a salt or a carbohydrate; or a combination of two or more of these. 4. Method according to claim 3, characterized in that the salt is a potassium salt or a sodium salt. 5. Method according to claim 1, characterized in that the silk proteins are concentrated by filtration. 6. Method according to claim 5, characterized in that the silk proteins are concentrated by membrane filtration. 7. Method according to claim 6, characterized by the fact that membrane filtration is tangential flow filtration. Method according to any one of claims 1 to 7, characterized in that it further comprises increasing the concentration of silk proteins in the silk dope. Method according to claim 8, characterized in that it comprises dialysis of the silk dope against a dehydration solution. 10. Method according to claim 9, characterized in that the dehydration solution comprises a hygroscopic polymer. 11. Method according to claim 10, characterized in that the hygroscopic polymer is selected from the group consisting of polyethylene glycol, amylase and sericin, or a combination of two or more of these. Method according to any one of claims 1 to 11, characterized in that the silk dope comprises at least 0.5% w / v of silk proteins. 13. Method according to claim 12, characterized in that the silk dope comprises 0.5% to 15% w / v of silk proteins. 14. Method according to any one of claims 1 to 13, characterized in that the cells are bacterial cells. 15. Method according to any one of claims 1 to 14, characterized in that step (i) further comprises isolating the inclusion bodies from the lysed cells. 16. Method according to any one of claims 1 to 15, characterized in that it further comprises culturing the cells before step (i). 17. Method according to any one of claims 1 to 16, characterized in that the portion of the silk protein that is capable of forming a tertiary structure comprising a coiled-coil structure, comprises at least 10 copies of the heptad abcdefg sequence, and in which at least 25% of the amino acids in the a and d positions are alanine residues. 18. Method according to any one of claims 1 to 17, characterized in that the silk proteins comprise a sequence selected from an amino acid sequence as provided in any of the IDS. SEQ. Nos 1, 3, 5, 7, 17, 19, 21, 23, 33, 35, 37, 39, 49, 51, 53, 55, 65, 67, 69, 71, 81, 83, 85, 87 or 97 . 19. Method according to any one of claims 1 to 18, characterized in that the silk proteins comprise a first silk protein comprising an amino acid sequence as provided in any of the IDS. SEQ. Nos. 1, 17, 33, 49, 65 or 81; a second silk protein comprising an amino acid sequence as provided in any of the IDS. SEQ. Nos. 3, 19, 35, 51, 67 or 83; a third silk protein comprising an amino acid sequence as provided in any of the IDS. SEQ. Nos. 5, 21, 37, 53, 69 or 85; and a fourth silk protein that comprises an amino acid sequence as provided in any of the IDS. SEQ. Nos 7, 23, 39, 55, 71 or 87. 20. Method according to claim 19, characterized in that the first silk protein, the second silk protein, the third silk protein and / or the fourth silk protein are produced by the same cells. 21. Method according to claim 19, characterized in that the first silk protein, the second silk protein, the third silk protein and / or the fourth silk protein are produced by different cells. 22. Method according to any one of claims 19 to 21, characterized in that step (ii) comprises equimolar amounts of the first silk protein, the second silk protein, the third silk protein and the fourth silk protein. 23. Method according to any one of claims 1 to 22, characterized in that the anionic surfactant is sodium dodecyl sulfate (SDS), ammonium lauryl sulfate or other alkyl sulfate salts, sodium 1-octane sulfonate monohydrate, lauroyl sodium sarcosinate, sodium lauryl ether sulfate (SLES), sodium taurodeoxycholate hydrate, alkyl benzene sulfonate, or a combination of two or more of these. 24. Method according to claim 23, characterized in that the anionic surfactant is sodium dodecyl sulfate (SDS). 25. Method for producing silk dope comprising silk proteins that have a coiled-coil tertiary structure, the method characterized by comprising: i) obtaining supernatant from cell cultures, or from a cellless expression system, producing one or more silk proteins capable of forming a coiled-coil tertiary structure, ii) solubilize the silk proteins by their contact with an amount of anionic surfactant sufficient to solubilize the silk proteins, and iii) concentrate the silk proteins while reducing the amount of anionic surfactant to produce the silk dope, in which the silk dope comprises the anionic surfactant and the silk proteins that have a coiled-coil tertiary structure, and in which the cells are bacterial cells, yeast cells, insect cells , plant cells or animal cells, or a combination of two or more of these. 26. Method according to claim 25, characterized in that step (i) further comprises increasing the concentration of silk proteins in the supernatant. 27. Method according to claim 26, characterized in that the concentration of silk proteins is increased by contacting the supernatant with an agent that precipitates silk proteins. 28. Method for producing a silk fiber characterized by comprising the extrusion and / or extraction of the silk dope produced by the method as defined in any one of claims 1 to 27. 29. Method according to claim 28, characterized in that the extrusion comprises the passage of the silk dope through a capillary tube from 5 μm to 500 μm. 30. The method of claim 28 or 29, characterized in that the silk dope is extruded into a solution comprising alcohol. 31. Method according to claim 30, characterized in that the alcohol is methanol and the concentration of methanol in the solution is 40% to 80% v / v. 32. Method for producing a silk film characterized in that it comprises molding the silk dope produced by the method as defined in any one of claims 1 to 27.

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